Inhibition of inflammation using interleukin-1β-converting enzyme (ICE)/CED-3 family inhibitors

ABSTRACT

The present invention provides methods for expanding and increasing survival of hematopoietic cell populations, for prolonging viability of an organ for transplantation, and enhancing bioproduction, using interleukin-1β-converting enzyme (ICE)/CED-3 family inhibitors. Exemplary compounds useful in the methods of the invention are provided herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to programmed cell death andspecifically to compositions and methods for ameliorating or preventinginflammation and symptoms associated therewith usinginterleukin-1β-converting enzyme (ICE)/CED-3 family inhibitors.

2. Description of the Related Art

The present invention relates generally to programmed cell death andspecifically to methods for expansion of hematopoietic cells, forprolonging viability of an organ for transplantation, and maintainingviability of cell lines used in bioproduction usinginterleukin-1β-converting enzyme (ICE)/CED-3 family inhibitors.

Necrosis and apoptosis are two basic processes by which cells may die.In necrosis cell death usually is a result of cell injury. The cellsgenerally swell and lyse, and the cell contents ultimately spill intothe extracellular space. By contrast, apoptosis is a mode of cell deathin which single cells are deleted in the midst of living tissues.Apoptosis accounts for most of the programmed cell death in tissueremodeling and for the cell loss that accompanies atrophy of adulttissues following withdrawal of endocrine and other growth stimuli. Inaddition, apoptosis is believed to be responsible for the physiologicdeath of cells in the course of normal tissue turnover (i.e., tissuehomeostasis) (Kerr, J. F. et al., Br. J. Cancer 26:239-257, 1972;Wyllie, A. H. et al., Int. Rev. Cytol. 68:251-306, 1980).

The effector mechanisms of apoptosis are not completely understood, butultimately, certain nuclear changes occur that appear to be caused bythe activation of endogenous nucleases that cleave chromatin betweennucleosomes and reduce the content of intact DNA in apoptotic cells. Anumber of regulators of apoptosis have been identified. Some of theseare already familiar as protooncogenes and oncosuppressor genes,including c-myc, bcl-2, p53, and ras. The protooncogene products andoncosuppressor proteins are believed to control cellular susceptibilityto apoptosis (Isaacs, J. T., Curr. Opin. Oncol. 6:82-89, 1994). C-myccan determine whether cells continuously proliferate or enter apoptosis,depending on the availability of critical growth factors (Bisonnette, R.P. et al., In Apoptosis II: The Molecular Basis of Apoptosis in Disease.Cold Spring Harbor Laboratory Press, 1994). In cultured cells,proliferation is usually observed in the presence of c-myc and growthfactors, whereas apoptosis is seen when c-myc is present but growthfactors are absent. Certain other oncogenes (e.g., bcl-2) rescue cellsfrom susceptibility to apoptosis. Specifically, members of the bcl-2gene family can act to inhibit programmed cell death (e.g., bcl-2,bcl-xL, ced-9) or promote cell death (e.g., bax, bak, bcl-xS).Additionally, members of the ICE/CED-3 family can promote cell death(e.g., ICE, CPP32, Ich-1, CED3).

Interleukin 1 (“IL-1”) is a major pro-inflammatory and immunoregulatoryprotein that stimulates fibroblast differentiation and proliferation,the production of prostaglandins, collagenase and phospholipase bysynovial cells and chondrocytes, basophil and eosinophil degranulationand neutrophil activation. (Oppenheim, J. H. et al., Immunology Today7:45-56, 1986). As such, it is involved in the pathogenesis of chronicand acute inflammatory and autoimmune diseases. IL-1 is predominantlyproduced by peripheral blood monocytes as part of the inflammatoryresponse. (Mosely, B. S. et al., Proc. Nat. Acad. Sci, 84:4572-4576,1987; Lonnemann, G. et al., Eur I. Immunol. 19:1531-1536, 1989).

Mammalian IL-1β is synthesized as a precursor polypeptide of about 31.5kDa (Linjuco, et al., Proc. Natl. Acad Sci. USA 83:3972, 1986).Precursor IL-1β is unable to bind to IL-1 receptors and is biologicallyinactive (Mosley et al., J. Biol. Chem. 262:2941, 1987). Biologicalactivity appears dependent upon proteolytic processing which results inthe conversion of the precursor 31.5 kDa form to the mature 17.5 kDaform.

Proteolytic maturation of human precursor IL-1β to mature, 17 kDa IL-1βresults from cleavage between Asp¹¹⁶ and Ala¹¹⁷. An endoproteinase,termed Interleukin-1β Converting Enzyme (ICE), has been identified inhuman monocytes that is capable of cleaving the IL-1β precursor atAsp¹¹⁶-Ala¹¹⁷, as well as at the site Asp²⁷-Gly²⁸, and generating matureIL-1β with the appropriate amino terminus at Ala¹¹⁷. The Asp at position116 has been found to be essential for cleavage, since substitution ofAla (Kostura et al., Proc. Natl. Acad Sci. 86:5227, 1989) or other aminoacids (Howard et al., J. Immunol. 147:2964, 1991) for Asp inhibits thiscleavage event.

The substrate specificity of human ICE has been defined with the use ofpeptides that span the cleavage site of the enzyme. Two features ofpeptide substrates are essential for catalytic recognition by theenzyme. First, there is a strong preference for aspartic acid adjacentto the cleavage site, in that any substitution of this residue in theIL-1β precursor and peptide substrates leads to a substantial reductionin the rate of catalysis (Kostura et al., Proc. Natl. Acad Sci. 86:5227,1989; Sleath et al., J. Biol. Chem. 265:14526, 1990; Howard et al., J.Immunol. 147:2964, 1991). There is an equally stringent requirement forfour amino acids to the left of the cleavage site, whereas methylamineis sufficient to the right. The minimal substrate for the enzyme,AC-Tyr-Val-Ala-Asp-NH—CH₃, is a particularly good peptide substrate witha relative Vmax/Km similar to that of the IL-1β precursor itself(Thomberry et al., Nature 356:768, 1992).

ICE is a cysteinyl proteinase by the following criteria: (1) thediazomethylketone AC-Tyr-Val-Ala-Asp-COCHN₂ is a potent, competitive,irreversible inhibitor of the enzyme, (2) inactivation of the enzyme byiodoacetate is competitive with substrate, and (3) the catalyticallyactive Cys reacts selectively with [¹⁴C] iodoacetate more than 10 timesfaster than do other cysteines or dithiothreitol (Thomberry et al.,Nature 356:768, 1992).

ICE is related structurally and functionally to the CED-3 protease thatfunctions as a cell death effector in the roundworm C. elegans (Yuan etal., Cell 75:641, 1993). ICE and CED-3 form part of a larger family ofproteases (the ICE/CED-3 family) that includes CPP32, ICH-1, Mch-2,ICE_(rel) II, ICE_(rel) III, Mch-3, Mch4 and Mch-5. All of these enzymesare cysteine proteases that share significant homology at their activesites. They also share the specificity for substrate cleavage at asp-xbonds. Additionally, each of the ICE/CED-3 family members is synthesizedas a pro-enzyme that is then proteolytically activated to form an activeenzyme.

Thus, disease states in which inhibitors of the ICE/ced-3 family ofcysteine proteases may be useful as therapeutic agents include:infectious diseases, such as meningitis and salpingitis; septic shock,respiratory diseases; inflammatory conditions, such as arthritis,cholangitis, colitis, encephalitis, endocerolitis, hepatitis,pancreatitis and reperfusion injury, ischemic diseases such as themyocardial infarction, stroke and ischemic kidney disease; immune-baseddiseases, such as hypersensitivity; auto-immune diseases, such asmultiple sclerosis; bone diseases; and certain neurodegenerativediseases.

In various cell culture systems, it has been shown that inhibition ofICE/CED-3 family members can effectively inhibit apoptosis. For example,the compound acetyl-DEVD-aldehyde inhibited anti-Fas induced apoptosisin a T-lymphocyte cell line (Schlegel et al., J. Biol. Chem. 271:1841,1996; Enari et al., Nature 380:723, 1996). Similarly,acetyl-YVAD-aldehyde and acetyl-YVAD-chloromethylketone blocked thedeath of motoneurons in vitro and in vivo (Milligan et al., Neuron15:385, 1995). In addition, the ICE/CED-3 family inhibitorBoc-D-(benzyl) chloromethylketone as well as crmA prevented the celldeath of mammary epithelial cells that occurs in the absence ofextracellular matrix (Boudreau et al., Science 267:891, 1995).

It is known that control of apoptosis may have utility in treatingdisease. Specifically, inhibitors of the ICE/CED-3 family may havetherapeutic effects. For example, it has been suggested that inhibitionof ICE may be useful in the treatment of inflammatory disorders (Dolleet al., J. Med. Chem. 37:563, 1994; Thomberry et al., Biochemistry33:3934, 1994). It is also known that inhibitors of ICE/CED-3 familymembers may have utility in treating degenerative diseases such asneurodegenerative diseases (e.g., Alzheimer's disease, Parkinson'sdisease, amyotrophic lateral sclerosis, Huntington's disease), ischemicdisease of heart or central nervous system (i.e., myocardial infarctionand stroke), and traumatic brain injury, as well as in alopecia, AIDSand toxin induced liver disease (Nicholson, Nature Biotechnology 14:297,1996).

Peptide and peptidyl inhibitors of ICE have been described. However,such inhibitors have been typically characterized by undesirablepharmacologic properties, such as poor oral absorption, poor stabilityand rapid metabolism. (Plattner, J. J. and D. W. Norbeck, Drug DiscoveryTechnologies, C. R. Clark and W. H. Moos, Eds. (Ellis Horwood,Chichester, England, 1990, pp. 92-126.) These undesirable propertieshave hampered their development into effective drugs. The methods ofthis invention include either the use of conformationally constraineddipeptide mimetics or a N-substituted indolyl peptide replacement. Thesemimetics exhibit improved properties relative to their peptidiccounterparts, for example, such as improved absorption and metabolicstability resulting in enhanced bioavailability.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to ameliorating inflammation and/orits symptoms, such as redness, irritation, itching, edema (swelling),burning, and etc. by inhibiting the activity of proteases of theinterleukin-1β-converting enzyme (ICE)/CED-3 family. The currentinvention provides new compositions and methods for using ICE/CED-3inhibitors.

The present invention generally provides methods and compositions forpreventing or treating inflammation. In one aspect, the presentinvention provides a method for preventing or treating inflammation bycontacting a cell population with an inhibiting effective amount of areagent that suppresses the protease activity of at least one member ofthe interleukin-1beta-converting enzyme (ICE)/CED-3 family, therebypreventing or treating inflammation. In certain embodiments, saidinflammation is chronic inflammation, acute inflammation, or due to aninflammatory disease. In a further embodiment, the inflammatory diseaseis selected from the group consisting of septic shock, septicemia, andadult respiratory distress syndrome. In other embodiments, the reagentsuppresses the protease activity in an irreversible manner or areversible manner. In another embodiment, the reagent is a compound or apharmaceutically acceptable salt thereof. The compound is represented byformula 1:

wherein n is 1 or 2; R¹ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl,(substituted)phenyl, phenylalkyl, (substituted)phenylalkyl, heteroaryl,(heteroaryl)alkyl or (CH₂)_(m)CO₂R⁴, wherein m=1-4, and R⁴ is as definedbelow; R² is a hydrogen atom, chloro, alkyl,cycloalkyl,(cycloalkyl)alkyl, phenyl,(substituted)phenyl, phenylalkyl,(substituted)phenylalkyl, heteroaryl, (heteroaryl)alkyl or(CH₂)_(p)CO₂R⁵, wherein p=0-4, and R⁵ is as defined below; R³ is ahydrogen atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or(substituted)phenylalkyl; R⁴ is a hydrogen atom, alkyl, cycloalkyl,(cycloalkyl)alkyl, phenylalkyl, or (substituted)phenylalkyl; R⁵ is ahydrogen atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or(substituted)phenylalkyl; A is a natural and unnatural amino acid; B isa hydrogen atom, a deuterium atom, alkyl, cycloalkyl, (cycloalkyl)alkyl,phenyl,(substituted)phenyl, phenylalkyl, (substituted)phenylalkyl,heteroaryl, (heteroaryl)alkyl, halomethyl, CH₂ZR⁶, CH₂OCO(aryl),CH₂OCO(heteroaryl), or CH₂OPO(R⁷)R⁸, wherein Z is an oxygen or a sulfuratom; R⁶ is phenyl, substituted phenyl, phenylalkyl, substitutedphenylalkyl, heteroaryl, or (heteroaryl)alkyl; R⁷ and R⁸ areindependently selected from a group consisting of alkyl, cycloalkyl,phenyl, substituted phenyl, phenylalkyl, (substituted phenyl) alkyl, and(cycloalkyl) alkyl; and X and Y are independently selected from thegroup consisting of a hydrogen atom, halo, trihalomethyl, amino,protected amino, an amino salt, mono-substituted amino, di-substitutedamino, carboxy, protected carboxy, a carboxylate salt, hydroxy,protected hydroxy, a salt of a hydroxy group, lower alkoxy, loweralkylthio, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl,(cycloalkyl)alkyl, substituted (cycloalkyl)alkyl, phenyl, substitutedphenyl, phenylalkyl, and (substituted phenyl)alkyl.

In yet another embodiment, the reagent is a compound of formula 3:

Wherein n is 1 or 2; m is 1 or 2; A is R²CO—, R³—O—CO—, or R⁴SO₂—; agroup of the formula:

further wherein R¹ is a hydrogen atom, alkyl or phenylalkyl; R² isalkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substitutedphenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; R³is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substitutedphenyl)alkyl; R⁴ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl,phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl,or (heteroaryl)alkyl; R⁵ is alkyl, cycloalkyl, (cycloalkyl)alkyl,phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl,heteroaryl, or (heteroaryl)alkyl; R⁶ is alkyl, cycloalkyl,(cycloalkyl)alkyl, phenylalkyl, or (substituted phenyl)alkyl; R⁷ isalkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substitutedphenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; R⁸is an amino acid side chain chosen from the group consisting of naturaland unnatural amino acids; B is a hydrogen atom, a deuterium atom,alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substitutedphenyl, (substituted phenyl)alkyl, heteroaryl, (heteroaryl)alkyl, orhalomethyl; a group of the formula:

—CH₂XR⁹;

wherein R⁹ is phenyl, substituted phenyl, phenylalkyl, (substitutedphenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; and X is an oxygen or asulfur atom; a group of the formula:

—CH₂—O—CO-(ARYL);

a group of the formula:

—CH₂—O—CO-(HETEROARYL);

a group of the formula:

—CH₂—O—PO(R¹⁰)R¹¹

wherein R¹⁰ and R¹¹ are independently selected from a group consistingof alkyl, cycloalkyl, phenyl, substituted phenyl, phenylalkyl and(substituted phenyl) alkyl; and the pharmaceutically-acceptable saltsthereof.

In another aspect, the present invention provides a compositioncomprising a cosmetic reagent that suppresses the protease activity ofat least one member of the interleukin-1beta-converting enzyme(ICE)/CED-3 family and a cosmetically or dermatologically acceptablecarrier, adapted for preventing or ameliorating irritation of the skinof a mammal due to said cosmetic. In certain embodiments, the reagentsuppresses the protease activity in an irreversible manner or reversiblemanner. In other embodiments, the reagent is a compound of formula 1 asdisclosed above. In yet other embodiments, the reagent is a compound offormula 3 as disclosed above.

In another aspect, the present invention provides a method forpreventing or ameliorating inflammation due to contact of the skin of amammal with an irritant by contacting the skin with a reagent thatsuppresses the protease activity of at least one member of theinterleukin-1beta-converting enzyme (ICE)/CED-3 family. In oneembodiment, the irritant is a chemical irritant. In further embodiments,the chemical irritant is a cosmetic one or one from a plant. In yetfurther embodiments, the plant is selected from the group consisting ofPoison Ivy, Poison Oak, and Poison Sumac. In another embodiment, theirritant is radiation. In one further embodiment, the radiation isultraviolet radiation. In yet other embodiments, the reagent suppressesthe protease activity in an irreversible manner or reversible manner. Instill other embodiments, the reagent is a compound represented byformula 1 or formula 3 as discussed above.

In another aspect, the present invention provides a composition thatcomprises a reagent that suppresses the protease activity of at leastone member of the interleukin-1beta-converting enzyme (ICE)/CED-3 familyformulated for topical administration for use in preventing orameliorating inflammation due to skin irritation. In one embodiment, theformulation is selected from a lotion, a cream, a gel, a liquid, asolid, or a semisolid. In another embodiment, the skin irritation is dueto contact of the skin with a chemical irritant. In a furtherembodiment, the chemical irritant is a cosmetic or an agent derived froma plant. In yet another embodiment, the irritant is radiation. Incertain embodiments, the irritation is due to an insect sting, an insectbite or tissue damage. In further embodiments, the tissue damage is dueto physical trauma or disease. In yet further embodiments, the tissue(Physical trauma or disease) damage is selected from the groupconsisting of a bum, a scrape, a cut, frostbite, and chemical injury. Incertain embodiments, the reagent suppresses the protease activity in anirreversible manner or reversible manner. In certain embodiments, thereagent is a compound represented by formula 1 or formula 3 as discussedabove.

In another aspect, the present invention provides a method forpreventing or ameliorating inflammation due to contact of a tissue of amammal with an irritant by contacting said tissue with a reagent thatsuppresses the protease activity of at least one member of theinterleukin-1beta-converting enzyme (ICE)/CED-3 family. In oneembodiment, the irritant is a chemical irritant. In further embodiments,the chemical irritant is a cosmetic or an agent from a plant. In yetfurther embodiments, the plant is selected from the group consisting ofPoison Ivy, Poison Oak, and Poison Sumac. In another embodiment, theirritant is radiation. In further embodiment, the radiation isultraviolet radiation. In yet another embodiment, the irritant is abacterium. In other embodiments, the reagent suppresses the proteaseactivity in an irreversible manner or reversible manner. In certainembodiments, the reagent is a compound represented by formula 1 orformula 3 as discussed above.

In another aspect, the present invention provides a method forpreventing or ameliorating inflammation associated with tissue damage bycontacting said tissue with a reagent that suppresses the proteaseactivity of at least one member of the interleukin-1beta-convertingenzyme (ICE)/CED-3 family. In certain embodiments, the tissue damage isdue to physical trauma, an autoimmune response, an infectious disease,chronic disease, spinal or brain trauma, an acid, a base, or radiation.In other embodiments, the reagent suppresses the protease activity in anirreversible manner or reversible manner. In yet other embodiments, thereagent is a compound represented by formula 1 or formula 3 as discussedabove.

In another aspect, the present invention provides a composition thatcomprises a reagent that suppresses the protease activity of at leastone member of the interleukin-1beta-converting enzyme (ICE)/CED-3 familyand a pharmaceutical, dermatological, or cosmetic carrier formulated fortopical application to the skin or mucus membrane of an animal. In oneembodiment, the composition ameliorates symptoms associated with aninflammatory response. In a further embodiment, the symptoms compriseitching, redness, or swelling. In another embodiment, the composition isuseful in decreasing loss of collagen or maintaining skin elasticity andappearance. In certain embodiments, the reagent suppresses the proteaseactivity in an irreversible manner or reversible manner. In yet certainembodiments, the reagent is a compound represented by formula 1 orformula 3 as discussed above.

In yet another aspect, the present invention provides a method forreducing inflammation of a tissue by contacting said tissue with aneffective amount of a reagent that suppresses the protease activity ofat least one member of the interleukin-1beta-converting enzyme(ICE)/CED-3 family, thereby reducing inflammation of said tissue. In oneembodiment, the tissue is skin. In a further embodiment, the tissueinflammation is due to trauma, sunburn, eczema, contact allergy,dermatitis, psoriasis, erysipelas, acne, ingrown nails, cuts, burns,insect bites, insect stings, or pruritus. In another embodiment, thetissue is mucosa. In a further embodiment, the tissue inflammation isdue to vaginitis, hemorrhoids, conjunctivitis, periodontitis, wisdomtooth eruption, teeth extraction, gingivitis, periodontal abscesses, orprosthesis.

Exemplary compounds useful as ICE/CED-3 inhibitors are also includedherein. Such compounds and methods of synthesis are described in theirentirety in co-pending U.S. patent application Ser. No. 09/482,813 filedJan. 13, 2000, Ser. No. 09/550,917 filed Apr. 17, 2000, Ser. No.09/747,317, filed Dec. 20, 2000, Ser. No. 09/908,969 filed Jul. 18,2001, U.S. Pat. No. 5,869,519 issued Feb. 9, 1999, U.S. Pat. No.5,877,197 issued Mar. 2, 1999, U.S. Pat. No. 6,184,244 issued Feb. 6,2001, U.S. Pat. No. 6,187,771 issued Feb. 13, 2001, U.S. Pat. No.6,197,750 issued Mar. 6, 2001, U.S. Pat. No. 6,242,422 issued Jun. 5,2001 and their respective continuations-in-part.

These and other aspects of the present invention will become apparentupon reference to the following detailed description and attacheddrawings. All references disclosed herein are hereby incorporated byreference in their entirety as if each was incorporated individually.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 sets forth the activity of the compounds in Formula A ininhibiting the activity of ICE and hCPP32 enzymes.

FIG. 2 illustrates the activity of the compounds in Formula B regardingrecombinant ICE, hCPP32, MCH2 and MCH5 enzymes.

FIG. 3 illustrates the activity of the compounds in Formula C regardingrecombinant ICE, CPP32, MCH2 and MCH5 enzymes.

FIG. 4 sets forth the activity of compounds in Formula D in inhibitingthe activity of ICE, CPP32, MCH2, MCH3 and MCH5 enzymes.

FIG. 5 sets forth the activity of Example 106 in inhibiting the activityof ICE, CPP32, MCH2 and MCH5.

FIG. 6 shows results derived from FACS analysis demonstrating the effectof ICE/CED-3 inhibitors on neutrophil survival as measured by DNAcontent (% hypodiploid).

FIG. 7 shows the effect of ICE/CED-3 inhibitors or neutrophil survivalas measured by the ability of live neutrophils to undergo oxidativeburst.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions and methods for amelioratinginflammation and/or symptoms associated therewith, by inhibition ofmembers of the ICE/CED-3 family. This would include not only inhibitorsof ICE/CED-3 enzymatic activity, but also any method that specificallyprevents the expression of ICE/CED-3 family encoding genes. Thus,antisense RNA or DNA comprised of nucleotide sequences complementary toICE/CED-3 family member genes and capable of inhibiting thetranscription or translation of the relevant proteins, expression ofdominant negative forms of the ICE/CED-3 proteases (e.g., mutantsengineered to replace the active site cysteine with another amino acid,like serine or alanine), or antibodies which bind to ICE/CED-3 familypolypeptides, are within the scope of the invention, as are smallmolecule inhibitors, including peptides.

Before describing the methods of the invention, several exemplarycompounds useful in the methods of the invention are described below:

wherein:

n is 1 or 2;

R¹ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, (substituted)phenyl,phenylalkyl, (substituted)phenylalkyl, heteroaryl, (heteroaryl)alkyl or(CH₂)_(m)CO₂R⁴, wherein

m=1-4, and R⁴ is as defined below;

R² is a hydrogen atom, chloro, alkyl, cycloalkyl,(cycloalkyl)alkyl,phenyl, (substituted)phenyl, phenylalkyl, (substituted)phenylalkyl,heteroaryl, (heteroaryl)alkyl or (CH₂)PCO₂R⁵, wherein p=0-4, and R⁵ isas defined below;

R³ is a hydrogen atom, alkyl, cycloalkyl, (cycloalkyl)alkyl,phenylalkyl, or (substituted)phenylalkyl;

R⁴ is a hydrogen atom, alkyl, cycloalkyl, (cycloalkyl)alkyl,phenylalkyl, or (substituted)phenylalkyl;

R⁵ is a hydrogen atom, alkyl, cycloalkyl, (cycloalkyl)alkyl,phenylalkyl, or (substituted)phenylalkyl;

A is a natural or unnatural amino acid;

B is a hydrogen atom, a deuterium atom, alkyl, cycloalkyl,(cycloalkyl)alkyl, phenyl, (substituted)phenyl, phenylalkyl,(substituted)phenylalkyl, heteroaryl, (heteroaryl)alkyl, halomethyl,CH₂ZR⁶, CH₂OCO(aryl), or CH₂OCO(heteroaryl), or CH₂OPO(R⁷)R⁸, where Z isan oxygen or a sulfur atom;

R⁶ is phenyl, substituted phenyl, phenylalkyl, (substitutedphenyl)alkyl, heteroaryl or (heteroaryl)alkyl; and

R⁷ and R⁸ are independently selected from a group consisting of alkyl,cycloalkyl, phenyl, substituted phenyl, phenylalkyl, (substitutedphenyl)alkyl and (cycloalkyl)alkyl; and

X and Y are independently selected from the group consisting of ahydrogen atom, halo, trihalomethyl, amino, protected amino, an aminosalt, mono-substituted amino, di-substituted amino, carboxy, protectedcarboxy, a carboxylate salt, hydroxy, protected hydroxy, a salt of ahydroxy group, lower alkoxy, lower alkylthio, alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, (cycloalkyl)alkyl, substituted(cycloalkyl)alkyl, phenyl, substituted phenyl, phenylalkyl, and(substituted phenyl)alkyl; or a pharmaceutically acceptable saltthereof.

As used in the above formula 1 and in formula 3 below, the term “alkyl”means a straight or branched C₁ to C₈ carbon chain such as methyl,ethyl, tert-butyl, iso-propyl, n-octyl, and the like.

The term “cycloalkyl” means a mono-, bi-, or tricyclic ring that iseither fully saturated or partially unsaturated. Examples of such a ringinclude cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,adamantyl, cyclooctyl, cis- or trans decalin, bicyclo[2.2.1]hept-2-ene,cyclohex-1-enyl, cyclopent-1-enyl, 1,4-cyclooctadienyl, and the like.

The term “(cycloalkyl)alkyl” means the above-defined alkyl groupsubstituted with one of the above cycloalkyl rings. Examples of such agroup include (cyclohexyl)methyl, 3-(cyclopropyl)-n-propyl,5-(cyclopentyl)hexyl, 6-(adamantyl)hexyl, and the like.

The term “substituted phenyl” specifies a phenyl group substituted withone or more, and preferably one or two, moieties chosen from the groupsconsisting of halogen, hydroxy, protected hydroxy, cyano, nitro,tifluoromethyl, C₁ to C₇ alkyl, C₁ to C₇ alkoxy, C₁ to C₇ acyl, C₁ to C₇acyloxy, carboxy, protected carboxy, carboxymethyl, protectedcarboxymethyl, hydroxymethyl, protected hydroxymethyl, amino, protectedamino, (monosubstituted)amino, protected (monosubstituted) amino,(disubstituted)amino, carboxamide, protected carboxamide, N—(C₁ to C₆alkyl)carboxamide, protected N—(C₁ to C₆ alkyl)carboxamide, N,N-di(C₁ toC₆ alkyl)carboxamide, N—((C to C₆ alkyl)sulfonyl)amino,N-(phenylsulfonyl)amino or by a substituted or unsubstituted phenylgroup, such that in the latter case a biphenyl or naphthyl groupresults.

Examples of the term “substituted phenyl” includes a mono- ordi(halo)phenyl group such as 2-, 3- or 4-chlorophenyl,2,6-dichlorophenyl, 2,5-dichlorophenyl, 3,4-dichlorophenyl, 2-, 3- or4-bromophenyl, 3,4-dibromophenyl, 3-chloro-4-fluorophenyl, 2-, 3- or4-fluorophenyl and the like; a mono or di(hydroxy)phenyl group such as2-, 3-, or 4-hydroxyphenyl, 2,4-dihydroxyphenyl, the protected-hydroxyderivatives thereof and the like; a nitrophenyl group such as 2-, 3-, or4-nitrophenyl; a cyanophenyl group, for example, 2-, 3- or4-cyanophenyl; a mono- or di(alkyl)phenyl group such as 2-, 3-, or4-methylphenyl, 2,4-dimethylphenyl, 2-, 3- or 4-(iso-propyl)phenyl, 2-,3-, or 4-ethylphenyl, 2-, 3- or 4-(n-propyl)phenyl and the like; a monoor di(alkoxy)phenyl group, for example, 2,6-dimethoxyphenyl, 2-, 3- or4-(iso-propoxy)phenyl, 2-, 3- or 4-(t-butoxy)phenyl,3-ethoxy-4-methoxyphenyl and the like; 2-, 3- or4-trifluoromethylphenyl; a mono- or dicarboxyphenyl or (protectedcarboxy)phenyl group such as 2-, 3- or 4-carboxyphenyl or2,4-di(protected carboxy)phenyl; a mono- or di(hydroxymethyl)phenyl or(protected hydroxymethyl)phenyl such as 2-, 3- or 4-(protectedhydroxymethyl)phenyl or 3,4-di(hydroxymethyl)phenyl; a mono- ordi(aminomethyl)phenyl or (protected aminomethyl)phenyl such as 2-, 3- or4-(aminomethyl)phenyl or 2,4-(protected aminomethyl)phenyl; or a mono-or di(N-(methylsulfonylamino))phenyl such as 2, 3 or4-(N-(methylsulfonylamino))phenyl. Also, the term “substituted phenyl”represents disubstituted phenyl groups wherein the substituents aredifferent, for example, 3-methyl-4-hydroxyphenyl,3-chloro-4-hydroxyphenyl, 2-methoxy-4-bromophenyl,4-ethyl-2-hydroxyphenyl, 3-hydroxy-4-nitrophenyl,2-hydroxy-4-chlorophenyl, and the like.

The term “(substituted phenyl)alkyl” means one of the above substitutedphenyl groups attached to one of the above-described alkyl groups.Examples of such groups include 2-phenyl-1-chloroethyl,2-(4′-methoxyphenyl)ethyl, 4-(2′,6′-dihydroxy phenyl)n-hexyl,2-(5′-cyano-3′-methoxyphenyl)n-pentyl, 3-(2′,6′dimethylphenyl)n-propyl,4-chloro-3-aminobenzyl, 6-(4′-methoxyphenyl)-3-carboxy(n-hexyl),5-(4′-aminomethylphenyl)-3-(aminomethyl)n-pentyl,5-phenyl-3-oxo-n-pent-1-yl, (4-hydroxynapth-2-yl)methyl, and the like.

The terms “halo” and “halogen” refer to the fluoro, chloro, bromo oriodo groups. There can be one or more halogen, which are the same ordifferent. Preferred halogens are chloro and fluoro.

The term “aryl” refers to aromatic five and six membered carbocyclicrings. Six membered rings are preferred.

The term “heteroaryl” denotes optionally substituted five-membered orsix-membered rings that have 1 to 4 heteroatoms, such as oxygen, sulfurand/or nitrogen atoms, in particular nitrogen, either alone or inconjunction with sulfur or oxygen ring atoms. These five-membered orsix-membered rings are fully unsaturated.

The following ring systems are examples of the heterocyclic (whethersubstituted or unsubstituted) radicals denoted by the term “heteroaryl”:thienyl, furyl, pyrrolyl, pyrrolidinyl, imidazolyl, isoxazolyl,triazolyl, thiadiazolyl, oxadiazolyl, tetrazolyl, thiatriazolyl,oxatriazolyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, oxazinyl,triazinyl, thiadiazinyl tetrazolo, 1,5-[b]pyridazinyl and purinyl, aswell as benzo-fused derivatives, for example, benzoxazolyl,benzothiazolyl, benzimidazolyl and indolyl.

Substituents for the above optionally substituted heteroaryl rings arefrom one to three halo, trihalomethyl, amino, protected amino, aminosalts, mono-substituted amino, di-substituted amino, carboxy, protectedcarboxy, carboxylate salts, hydroxy, protected hydroxy, salts of ahydroxy group, lower alkoxy, lower alkylthio, alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, (cycloalkyl)alkyl, substituted(cycloalkyl)alkyl, phenyl, substituted phenyl, phenylalkyl, and(substituted phenyl)alkyl groups. Substituents for the heteroaryl groupare as heretofore defined, or as set forth below. As used in conjunctionwith the above substituents for heteroaryl rings, “trihalomethyl” can betrifluoromethyl, trichloromethyl, tribromomethyl or triiodomethyl,“lower alkoxy” means a C, to C₄ alkoxy group, similarly, “loweralkylthio” means a C₁ to C₄ alkylthio group. The term “substitutedalkyl” means the above-defined alkyl group substituted from one to threetimes by a hydroxy, protected hydroxy, amino, protected amino, cyano,halo, trifluoromethyl, mono-substituted amino, di-substituted amino,lower alkoxy, lower alkylthio, carboxy, protected carboxy, or a carboxy,amino, and/or hydroxy salt. As used in conjunction with the substituentsfor the heteroaryl rings, the terms “substituted (cycloalkyl)alkyl” and“substituted cycloalkyl” are as defined above substituted with the samegroups as listed for a “substituted alkyl” group. The term“(monosubstituted)amino” refers to an amino group with one substituentchosen from the group consisting of phenyl, substituted phenyl, alkyl,substituted alkyl, C₁ to C₇ acyl, C₂ to C₇ alkenyl, C₂ to C₇ substitutedalkenyl, C₂ to C₇ alkynyl, C₇ to C₁₆ alkylaryl, C₇ to C₁₆ substitutedalkylaryl and heteroaryl group. The (monosubstituted)amino canadditionally have an amino-protecting group as encompassed by the term“protected (monosubstituted)amino.” The term “(disubstituted)amino”refers to amino groups with two substituents chosen from the groupconsisting of phenyl, substituted phenyl, alkyl, substituted alkyl, C₁to C₇ acyl, C₂ to C₇ alkenyl, C₂ to C₇ alkynyl, C₇ to C₁₆ alkylaryl, C₇to C₁₆ substituted alkylaryl and heteroaryl. The two substituents can bethe same or different. The term “heteroaryl(alkyl)” denotes an alkylgroup as defined above, substituted at any position by a heteroarylgroup, as above defined.

Furthermore, the above optionally substituted five-membered orsix-membered heterocyclic rings can optionally be fused to a aromatic5-membered or 6-membered aryl or heteroaryl ring system. For example,the rings can be optionally fused to an aromatic 5-membered or6-membered ring system such as a pyridine or a triazole system, andpreferably to a benzene ring.

The term “pharmaceutically-acceptable salt” encompasses those salts thatform with the carboxylate anions and includes salts formed with theorganic and inorganic cations such as those chosen from the alkali andalkaline earth metals, (for example, lithium, sodium, potassium,magnesium, barium and calcium); and ammonium ion; and the organiccations (for example, dibenzylammonium, benzylammonium,2-hydroxyethylammonium, bis(2-hydroxyethyl)ammonium,phenylethylbenzylammonium, dibenzylethylenediammonium, and likecations.) Other cations encompassed by the above term include theprotonated form of procaine, quinine and N-methylglucosamine, theprotonated forms of basic amino acids such as glycine, ornithine,histidine, phenylglycine, lysine, and arginine. Furthermore, anyzwitterionic form of the instant compounds formed by a carboxylic acidand an amino group is referred to by this term. A preferred cation forthe carboxylate anion is the sodium cation. Furthermore, the termincludes salts that form by standard acid-base reactions with basicgroups (such as amino groups) and includes organic or inorganic acids.Such acids include hydrochloric, sulfuric, phosphoric, acetic, succinic,citric, lactic, maleic, fumaric, palmitic, cholic, pamoic, mucic,D-glutamic, D-camphoric, glutaric, phthalic, tartaric, lauric, stearic,salicyclic, methanesulfonic, benzenesulfonic, sorbic, picric, benzoic,cinnamic, and the like acids.

The compounds of Formula 1 may also exist as solvates and hydrates.Thus, these compounds may crystallize with, for example, waters ofhydration, or one, a number of, or any fraction thereof of molecules ofthe mother liquor solvent. The solvates and hydrates of such compoundsare included within the scope of this invention.

The term “carboxy-protecting group” as used herein refers to one of theester derivatives of the carboxylic acid group commonly employed toblock or protect the carboxylic acid group while reactions are carriedout on other functional groups on the compound. Examples of suchcarboxylic acid protecting groups include t-butyl, 4-nitrobenzyl,4-methoxybenzyl, 3,4-dimethoxybenzyl, 2,4-dimethoxybenzyl,2,4,6-trimethoxybenzyl, 2,4,6-trimethylbenzyl, pentamethylbenzyl,3,4-methylenedioxybenzyl, benzhydryl, 4,4′-dimethoxytrityl,4,4′,4″-trimethoxytrityl, 2-phenylpropyl, trimethylsilyl,t-butyldimethylsilyl, phenacyl, 2,2,2-trichloroethyl, β-(trimethylsilyl)ethyl, β-(di(n-butyl)methylsilyl) ethyl, p-toluenesulfonylethyl,4-nitrobenzylsulfonylethyl, allyl, cinnamyl,1-(trimethylsilylmethyl)-propenyl and like moieties. The species ofcarboxy-protecting group employed is not critical so long as thederivatized carboxylic acid is stable to the conditions of subsequentreaction(s) and can be removed at the appropriate point withoutdisrupting the remainder of the molecule. Further examples of thesegroups are found in C. B. Reese and E. Haslam, “Protective Groups inOrganic Chemistry,” J. G. W. McOmie, Ed., Plenum Press, New York, N.Y.,1973, Chapter 5, respectively, and T. W. Greene and P. G. M. Wuts,“Protective Groups in Organic Synthesis,” 2nd ed., John Wiley and Sons,New York, N.Y., 1991, Chapter 5, each of which is incorporated herein byreference. A related term is “protected carboxy,” which refers to acarboxy group substituted with one of the above carboxy-protectinggroups.

The term “hydroxy-protecting group” refers to readily cleavable groupsbonded to hydroxyl groups, such as the tetrahydropyranyl,2-methoxyprop-2-yl, 1-ethoxyeth-1-yl, methoxymethyl,β-methoxyethoxymethyl, methylthiomethyl, t-butyl, t-amyl, trityl,4-methoxytrityl, 4,4′-dimethoxytrityl, 4,4′,4″-trimethoxytrityl, benzyl,allyl, trimethylsilyl, (t-butyl)dimethylsilyl,2,2,2-trichloroethoxycarbonyl, and the like.

Further examples of hydroxy-protecting groups are described by C. B.Reese and E. Haslam, “Protective Groups in Organic Chemistry,” J. G. W.McOmie, Ed., Plenum Press, New York, N.Y., 1973, Chapters 3 and 4,respectively, and T. W. Greene and P. G. M. Wuts, “Protective Groups inOrganic Synthesis,” Second Edition, John Wiley and Sons, New York, N.Y.,1991, Chapters 2 and 3. A preferred hydroxy-protecting group is thetert-butyl group.

The related term “protected hydroxy” denotes a hydroxy group bonded toone of the above hydroxy-protecting groups.

The term “amino-protecting group” as used herein refers to substituentsof the amino group commonly employed to block or protect the aminofunctionality while reacting other functional groups of the molecule.The term “protected (monosubstituted)amino” means there is anamino-protecting group on the monosubstituted amino nitrogen atom.

Examples of such amino-protecting groups include the formyl (“For”)group, the trityl group, the phthalimido group, the trichloroacetylgroup, the trifluoroacetyl group, the chloroacetyl, bromoacetyl, andiodoacetyl groups, urethane-type protecting groups, such ast-butoxycarbonyl (“Boc”), 2-(4-biphenylyl)propyl-2-oxycarbonyl (“Bpoc”),2-phenylpropyl-2-oxycarbonyl (“Poc”), 2-(4-xenyl)isopropoxycarbonyl,1,1-diphenylethyl-1-oxycarbonyl, 1,1-diphenylpropyl-1-oxycarbonyl,2-(3,5-dimethoxyphenyl)propyl-2-oxycarbonyl (“Ddz”),2-H-toluyl)propyl-2-oxycarbonyl, cyclopentanyloxycarbonyl,1-methylcyclopentanyloxycarbonyl, cyclohexanyloxy-carbonyl,1-methyl-cyclohexanyloxycarbonyl, 2-methylcyclohexanyloxycarbonyl,2-(4-toluylsulfonyl)ethoxycarbonyl, 2-(methylsulfonyl)ethoxycarbonyl,2-(triphenylphosphino)ethoxycarbonyl, 9-fluorenylmethoxycarbonyl(“Fmoc”), 2-(trimethylsilyl)ethoxycarbonyl, allyloxycarbonyl,1-(trimethylsilylmethyl)prop-1-enyloxycarbonyl,5-benzisoxalylmethoxycarbonyl, 4-acetoxybenzyl-oxycarbonyl,2,2,2-trichloroethoxycarbonyl, 2-ethynyl-2-propoxycarbonyl,cyclopropylmethoxycarbonyl, isobomyloxycarbonyl, 1-piperidyloxycarbonyl,benzyloxycarbonyl (“Cbz”), 4-phenylbenzyloxycarbonyl,2-methylbenzyloxycarbonyl, α-2,4,5, -tetramethylbenzyl-oxycarbonyl(“Tmz”), 4-methoxybenzyloxycarbonyl, 4-fluorobenzyloxycarbonyl,4-chlorobenzyloxycarbonyl, 3-chlorobenzyloxycarbonyl,2-chlorobenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl,4-bromobenzyloxycarbonyl, 3-bromobenzyloxycarbonyl,4-nitrobenzyloxycarbonyl, 4-cyanobenzyloxycarbonyl,4-(decyloxy)benzyloxycarbonyl and the like; the benzoyhnethylsulfonylgroup, the 2,2,5,7,8-pentamethylchroman-6-sulfonyl group (“PMC”), thedithiasuccinoyl (“Dts”) group, the 2-(nitro)phenyl-sulfenyl group(“Nps”), the diphenylphosphine oxide group, and like amino-protectinggroups. The species of amino-protecting group employed is not criticalso long as the derivatized amino group is stable to the conditions ofthe subsequent reaction(s) and can be removed at the appropriate pointwithout disrupting the remainder of the molecule. Preferredamino-protecting groups are Boc, Cbz and Fmoc. Further examples ofamino-protecting groups embraced by the above term are well known inorganic synthesis and the peptide art and are described by, for example,T. W. Greene and P. G. M. Wuts, “Protective Groups in OrganicSynthesis,” 2nd Ed., John Wiley and Sons, New York, N.Y., 1991, Chapter7, M. Bodanzsky, “Principles of Peptide Synthesis,” 1st and 2nd revisedEd., Springer-Verlag, New York, N.Y., 1984 and 1993, and J. M. Stewartand J. D. Young, “Solid Phase Peptide Synthesis,” 2nd Ed., PierceChemical Co., Rockford, Ill., 1984, E. Atherton and R. C. Shephard,“Solid Phase Peptide Synthesis—A Practical Approach” IRL Press, Oxford,England (1989), each of which is incorporated herein by reference. Therelated term “protected amino” defines an amino group substituted withan amino-protecting group discussed above.

The terms “natural and unnatural amino acid” refers to both thenaturally occurring amino acids and other non-proteinogenic a-aminoacids commonly utilized by those in the peptide chemistry arts whenpreparing synthetic analogues of naturally occurring peptides, includingD and L forms. The naturally occurring amino acids are glycine, alanine,valine, leucine, isoleucine, serine, methionine, threonine,phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine,aspartic acid, asparagine, glutamic acid, glutamine, γ-carboxyglutamicacid, arginine, omithine and lysine. Examples of unnatural alpha-aminoacids include hydroxylysine, citrulline, kynurenine,(4-aminophenyl)alanine, 3-(2′-naphthyl)alanine, 3-(1′-naphthyl)alanine,methionine sulfone, (t-butyl)alanine, (t-butyl)glycine,4-hydroxyphenyl-glycine, aminoalanine, phenylglycine, vinylalanine,propargyl-glycine, 1,2,4-triazolo-3-alanine, thyronine,6-hydroxytryptophan, 5-hydroxytptophan, 3-hydroxy-kynurenine,3-aminotyrosine, trifluoromethylalanine, 2-thienylalanine,(2-(4-pyridyl)ethyl)cysteine, 3,4-dimethoxy-phenylalanine,3-(2′-thiazolyl)alanine, ibotenic acid,1-amino-1-cyclopentane-carboxylic acid, 1-amino-1-cyclohexanecarboxylicacid, quisqualic acid, 3-(trifluoromethylphenyl)alanine,(cyclohexyl)glycine, thiohistidine, 3-methoxytyrosine, norleucine,norvaline, alloisoleucine, homoarginine, thioproline, dehydro-proline,hydroxyproline, homoproline, indoline-2-carboxylic acid,1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid,1,2,3,4-tetrahydroquinoline-2-carboxylic acid, α-amino-n-butyric acid,cyclohexylalanine, 2-amino-3-phenylbutric acid, phenylalaninesubstituted at the ortho, meta, or para position of the phenyl moietywith one or two of the following groups: a (C₁ to C₄)alkyl, a (C₁ toC₄)alkoxy, a halogen or a nitro group, or substituted once with amethylenedioxy group; β-2- and 3-thienylalanine; β-2- and3-furanylalanine; P-2-, 3- and 4-pyridylalanine; β-(benzothienyl-2- and3-yl)alanine; β-(1- and 2-naphthyl)alanine; O-alkylated derivatives ofserine, threonine or tyrosine; S-alkylated cysteine, S-alkylatedhomocysteine, the O-sulfate, O-phosphate and O-carboxylate esters oftyrosine; 3-(sulfo)tyrosine, 3-(carboxy)tyrosine, 3-(phospho)tyrosine,the 4-methanesulfonic acid ester of tyrosine, 4-methanephosphonic acidester of tyrosine, 3,5-diiodotyrosine, 3-nitrotyrosine, ε-alkyllysine,and delta-alkyl ornithine. Any of these α-amino acids may be substitutedwith a methyl group at the alpha position, a halogen at any position ofthe aromatic residue on the a-amino side chain, or an appropriateprotective group at the O, N, or S atoms of the side chain residues.Appropriate protective groups are discussed above.

Depending on the choice of solvent and other conditions known to thepractitioner skilled in the art, compounds of this invention may alsotake the ketal or acetal form, which forms are included in the instantinvention.

In addition, it should be understood that the equilibrium forms of thecompounds of this invention may include tautomeric forms. All such formsof these compounds are expressly included in the present invention.

The compounds useful in the methods of the invention may be modified byappropriate functionalities to enhance selective biological properties.Such modifications are known in the art and include those which increasebiological penetration into a given biological system (e.g., blood,lymphatic system, central nervous system), increase oral availability,increase solubility to allow administration by injection, altermetabolism and alter rate of exertion. In addition, the compounds may bealtered to pro-drug form such that the desired compound is created inthe body of the patient as the result of the action of metabolic orother biochemical processes on the pro-drug. Some examples of pro-drugforms include ketal, acetal, oxime, and hydrazone forms of compoundswhich contain ketone or aldehyde groups, especially where they occur inthe group donated as “A” in Formula I or the modified aspartic orglutamic residues attached to the group denoted as “A”.

In the above Formula 1 or in Formula 3 below, a group of optimalcompounds occurs when n is one, more so when B is a hydrogen atom, andespecially so when R³ is a hydrogen atom or a t-butyl group. Of notewithin this group of compounds as those when A is naturally-occurringamino acid. This latter group of compounds will be referred to herein asthe “4-oxobutanoic compounds”.

Within this group of 4-oxobutanoic compounds is a group of optimalcompounds wherein R¹ is a methyl group, that is, the N-methylindolecompounds. One embodiment of this group of N-methylindole compoundsoccurs when A is an alanine, valine, leucine, phenylalanine, glycine ora proline residue. Compounds of note within each one of these groups ofnatural amino acid, N-methylindole compounds occur when theN-methylindole is otherwise unsubstituted, that is, wherein X, Y and R²are each a hydrogen atom, and optimally so when R³ is a hydrogen atom.

Another optimal group of 4-oxobutanoic compounds consists of theN-benzylindole compounds. For example, one group of the N-benzylindolecompounds occurs when A is an alanine residue. Of note within this groupof alanine compounds are those in which X, Y and R² are each a hydrogenatom, and especially so where R is a hydrogen atom.

An alternate optimal group of 4-oxobutanoic compounds occurs when theN-substituent of the indole group is a 1-butenyl group. An embodiment ofthis group of N-(1-butenyl)indole compounds occurs when A is a valineresidue, and especially so when X, Y and R² are each a hydrogen atom. Anoptimal group of this latter group of compounds occurs when R³ is ahydrogen atom.

Yet another group of optimal 4-oxobutanoic compounds occurs when theN-substituent of the indole ring is a 2′-acetic acid residue. Anexemplary group of the N-(2′-acetic acid compounds) occurs when A is analanine residue. An embodiment of this particular group of alaninecompounds occurs when X, Y and R² are each a hydrogen atom, andespecially so when R³ is a hydrogen atom.

A group of the 4-oxobutanoic compounds when the indole group issubstituted on the nitrogen with 3′-propionic acid residue is anotherexample of this invention. An optimal group of such N-(propionicacid)indole compounds occurs when A is an alanine residue. Of notewithin this group of alanine compounds are those when X, Y and R² areeach a hydrogen atom, and especially so when R³ is a hydrogen atom.

Another optimal group of compounds of Formula 1 occurs wherein n is oneand more so when B is a monofluoromethyl group. An embodiment of thesemonofluoromethyl compounds occurs when R³ is a hydrogen atom or at-butyl group, and an even more so when A is a natural amino acid. Anexample of these compounds wherein A is a natural amino acid occurs whenA is a valine residue. This latter group of valine compounds will bereferred to herein as the “4-oxo-5-(fluoropentanoic acid) compounds”.

One optimal group of 4-oxo-5-(fluoropentanoic acid) compounds occurswhen R¹ is a methyl group, in other words, the N-methylindole compounds.An exemplary group of such N-methylindole compounds occurs when R² is amethyl group and X and Y are each a hydrogen atom, and especially sowhen R³ is a hydrogen atom. Another exemplary group of suchN-methylindole compounds occurs when R² is a chloro atom and X and Y areeach a hydrogen atom, and especially so when R³ is a hydrogen atom. Athird exemplary group of N-methylindole compounds occurs when R² is achloro group, X is a 5-fluoro group, and Y is a hydrogen atom, andespecially so when R³ is a hydrogen atom.

Another optimal group of 4-oxo-5-(fluoro-pentanoic acid) compounds iscomposed of N-(3′-phenylprop-1-yl)indole compounds. A group of notewithin this latter class of compounds occurs when R², X and Y are each ahydrogen atom, and especially so when R is a hydrogen atom.

A third optimal group of 4-oxo-5-(fluoro-pentanoic acid) compounds hasan N-(carboxymethyl or protected carboxymethyl)indole moiety. Anembodiment of this group occurs wherein R², X and Y are each a hydrogenatom, and especially so wherein R is a hydrogen atom and the nitrogenatom of the indole ring is substituted with a carboxymethyl group.

Another optimal class of compounds of Formula 1 occurs when n is one andB is a (2,6-dichlorobenzyloxy)-methyl group and especially so when R³ isa hydrogen atom or a t-butyl group, and when A is a natural amino acid.An example of such a compounds occurs when R¹ is a methyl group andespecially so when R² is a methyl group.

The compounds of Formula 1 may be synthesized using conventionaltechniques as discussed below. Advantageously, these compounds areconveniently synthesized from readily available starting materials.

One synthetic route for synthesizing compounds is set forth in thefollowing Scheme 1:

In the above Scheme I, Formula (2), that is H2N-(Glu, Asp), is amodified aspartic or glutamic acid residue of Formulas 2a through 2d:

In the above Scheme I, (P) stands for an amino protecting group and (A)stands for a natural or unnatural amino acid, as discussed above.

The modified aspartic or glutamic acids of Formula 2a-d can be preparedby methods well known in the art. See, for example, European PatentApplication 519,748; PCT Patent Application No. PCT/EP92/02472; PCTPatent Application No. PCT/US91/06595; PCT Patent Application No.PCT/US91/02339; European Patent Application No. 623,592; World PatentApplication No. WO 93/09135; PCT Patent Application No. PCT/US94/08868;European Patent Application No. 623,606; European Patent Application No.618,223; European Patent Application No. 533,226; European PatentApplication No. 528,487; European Patent Application No. 618,233; PCTPatent Application No. PCT/EP92/02472; World Patent Application No. WO93/09135; PCT Patent Application No. PCT/US93/03589; and PCT PatentApplication No. PCT/US93/0048 1, all of which are herein incorporated byreference.

The coupling reactions carried out under Step A are performed in thepresence of a standard peptide coupling agent such as the combination ofthe combination of dicyclohexylcarbodiimide(DCC) and1-hydroxy-benzotriazole(HOBt), as well as the BOP(benzotriazolyloxy-trio-(dimethylamino)phosphonium hexafluorophosphate)reagent, pyBOP(benzotriazolyloxy-tris(N-pyrolidinyl)phosphoniumhexafluorophosphate),HBTU (O-benzotriazolyly-tetramethylisouronium-hexafluorophosphate), andEEDQ (1-ethyloxycarbonyl-2-ethyloxy-1,2-dihydroquinoline) reagents, thecombination of 1-ethyl(3,3′-dimethyl-1′-aminopropyl)carbodiimide (EDAC)and HOBt, and the like, as discussed in J. Jones, “Amino Acid andPeptide Synthesis,” Steven G. Davis ed., Oxford University Press,Oxford, pp. 25-41 (1992); M. Bodanzky, “Principles of PeptideSynthesis,” Hafner et al. ed., Springer-Verlag, Berlin Heidelberg, pp.9-52 and pp. 202-251 (1984); M. Bodanzky, “Peptide Chemistry, APractical Textbook,” Springer-Verlag, Berlin Heidelberg, pp. 55-73 andpp. 129-180; and Stewart and Young, “Solid Phase Peptide Synthesis,”Pierce Chemical Company, (1984), all of which are herein incorporated byreference. The amino protecting group is then removed and the resultingamine is coupled to the 2-(carboxy)indole of (3) (Step B). Again, thiscoupling reaction uses the standard peptide coupling reactions mentionedabove. The indole ring of (3) can be substituted before the reaction inStep B or afterwards. The synthesis and substitution reactions of suchan indole ring is well known, as is described, for example, in Brown, R.T. and Joule, J. A. in “Heterocyclic chemistry (ed. P. G. Sammes) (Vol.4 of Comprehensive Organic Chemistry, ed. D. Barton and W. D. Ollis),(1979), Pergamon Press, Oxford; Houlihan, W. J., (ed.) in “Indoles (TheChemistry of Heterocyclic Compounds,” [ed. A. Weissburger and E. C.Taylor], Vol. 25, Parts 1-3), Wiley Interscience, New York (1972); andSaxton, J. E. (ed.) in “Indoles (The Chemistry of HeterocyclicCompounds),” [ed. A. Weissburger and E. C. Taylor], Vol. 25, Part 4),Wiley Interscience, New York, (1979); all of which are incorporatedherewith by reference.

In the case where the coupling reaction was carried out with the aminoalcohol of Formula 2c, the alcohol moiety must be oxidized to thecorresponding carbonyl compound prior to removal of the protectinggroups. Preferred methods for the oxidation reaction include Swemoxidation (oxalyl chloride-dimethyl sulfoxide, methylene chloride at−78° C. followed by triethylamine); and Dess-Martin oxidation(Dess-Martin periodinane, t-butanol, and methylene chloride.) Theprotecting groups contained in substructures of the Formula 2a-d and Aare removed by methods well known in the art. These reactions andremoval of some or all of the protecting groups are involved in Step Cin the above Scheme.

The compounds of Formula 3, below, are also useful in the methods of theinvention:

wherein:

n is 1 or 2;

m is 1 or 2;

A is R² CO—, R³—O—CO—, or R⁴ SO₂—;

a group of the formula:

further wherein:

R¹ is a hydrogen atom, alkyl or phenylalkyl;

R² is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl,substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or(heteroaryl)alkyl;

R³ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substitutedphenyl)alkyl;

R⁴ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl,substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or(heteroaryl)alkyl;

R⁵ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl,substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or(heteroaryl)alkyl;

R⁶ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substitutedphenyl)alkyl;

R⁷ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl,substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or(heteroaryl)alkyl;

R⁸ is an amino acid side chain chosen from the group consisting ofnatural and unnatural amino acids;

B is a hydrogen atom, a deuterium atom, alkyl, cycloalkyl,(cycloalkyl)alkyl, phenyl, phenylalkyl, (substituted)phenyl,(substituted)phenylalkyl, heteroaryl, (heteroaryl)allyl, or halomethyl;

a group of the formula

—CH₂XR⁹;

wherein R⁹ is phenyl, phenylalkyl, substituted phenyl, (substitutedphenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; and X is an oxygen or asulfur atom;

a group of the formula:

—CH₂—O—CO-(aryl);

a group of the formula:

—CH₂—O—CO-(heteroaryl);

a group of the formula:

—CH₂—O—PO(R¹⁰)R¹¹

wherein R¹⁰ and R¹¹ are independently selected from a group consistingof alkyl, cycloalkyl, phenyl, substituted phenyl, phenylalkyl and(substituted phenyl) alkyl; and the pharmaceutically-acceptable saltsthereof.

The compounds of Formula 3 may also exist as solvates and hydrates.Thus, these compounds may crystallize with, for example, waters ofhydration, or one, a number of, or any fraction thereof of molecules ofthe mother liquor solvent. The solvates and hydrates of such compoundsare included within the scope of this invention.

The compounds of Formulas 1 and 3 of this invention may be synthesizedusing conventional techniques. Advantageously, these compounds areconveniently synthesized from readily available starting materials.

Thus, compounds of Formula 3 can be synthesized in general by combininga tricyclic nucleus set forth below in Formula 4:

with the modified aspartic and glutamic acid residues of Formula 5a-d:

in the presence of a standard peptide coupling agents such asdicyclohexylcarbodiimide(DCC)-1-hydroxybenzotriazole(HOBt), BOP reagent,pyBOP, TBTU, EEDQ,1-ethyl(3,3′-dimethyl-1′-aminopropyl)carbodiimide(EDAC)-HOBt, and thelike, as discussed in J. Jones, “Amino Acid and Peptide Synthesis,”Steven G. Davis ed., Oxford University Press, Oxford, pp. 25-41 (1992),herein incorporated by reference. In the above formula, A is an aminoprotecting group. The amino protecting group is then removed and theresulting amine is combined with the substituted acyl group of Formula6:

R^(c)—CO—X  (6)

or the sulfonyl group of Formula 7:

R⁴SO₂—X  (7)

In the above formulas, R¹ is as defined above, and R^(c) is R², R³—O,R⁴, or any of the side chains containing R⁸ as defined for group A inFormula 3. Of course, such moieties would have any hydroxy, carboxy oramino groups in the protected form so as not to interfere with thecoupling reaction (Formula 5a-d), the acylation reaction (Formula 4) orthe sulfonation reaction (Formula 7). X in the above Formulas representsa facile leaving group for the acylation or sulfonation reactions.

In the case where the coupling reaction was carried out with the aminoalcohol of Formula 5c, the alcohol moiety must be oxidized to thecorresponding carbonyl compound prior to removal of the protectinggroups. Preferred methods for the oxidation reaction include Swemoxidation (oxalyl chloride-dimethyl sulfoxide, methylene chloride at−78° C. followed by triethylmine; and Dess-Martin oxidation (Dess-Martinperiodinane, t-butanol, and methylene chloride.) The protecting groupscontained in substructures of the Formula 5a-d and A are removed bymethods well known in the art.

The tricyclic nucleus of Formula 3 is synthesized by methods known inthe art. For example, see D. S. Karanewsky, U.S. Pat. No. 5,504,080issued Apr. 2, 1996; J. A. Robl et al., Tetrahedron Letters 36:1593-1596(1995); and S. De Lombaert et al., Tetrahedron Letters 35:7513-7516(1994), all of which are incorporated herein by reference.

The modified aspartic or glutamic acid for Formula 5a-d can beelaborated by methods well known in the art. See, for example, EuropeanPatent Application 519,748; PCT Patent Application No. PCT/EP92/02472;PCT Patent Application No. PCT/US91/06595; PCT Patent Application No.PCT/US91/02339; European Patent Application No. 623,592; World PatentApplication No. WO 93/09135; PCT Patent Application No. PCT/US94/08868;European Patent Application No. 623,606; European Patent Application No.618,223; European Patent Application No. 533,226; European PatentApplication No. 528,487; European Patent Application No. 618,233; PCTPatent Application No. PCT/EP92/02472; World Patent Application No. WO93/09135; PCT Patent Application No. PCT/US93/03589; and PCT PatentApplication No. PCT/US93/00481, all of which are herein incorporated byreference.

The acyl group of Formula 6 and the corresponding R⁴SO₂ groups are alsosynthesized by methods well known in the art. See, for example, U.S.Pat. No. 5,504,080, issued Apr. 2, 1996, herein incorporated byreference. While this group can be elaborated once bonded to thetricyclic nucleus, it is preferable that it be intact before beingattached to the nucleus.

Once the side chains of Formula 5 and Formula 6 or Formula 7 are bondedto the tricyclic nucleus of Formula 3, one skilled in the art wouldusually remove any amino, hydroxy, or carboxy-protecting groups toenhance the activity of the synthesized molecule.

A “dermatologic agent,” as used herein, refers to an agent for thetreatment of dermatologic disorders of a human or animal, including butnot limited to acne vulgaris, gential herpes, psoriasis, eczema, tineapedis and the like.

A “cosmetic agent,” as used herein, refers to an agent that is intendedto be rubbed, poured, sprinkled, or sprayed on, introduced onto, orotherwise applied to the human body or any part thereof for cleaning,beautifying, promoting attractiveness, or altering the appearancethereof.

As used herein, compositions for cosmetic use, dermatological use, fortopical use, or skin applications, encompasses treatments and uses whichare specifically medicinal as wells as conventional cosmetic uses suchas beauty aids and toiletries.

The term “topical” as employed in this application relates to theintroduction of formulations of the invention, incorporated in asuitable base or vehicle, at the site of the area for the exertion oflocal action. Accordingly, such topical compositions include those formsin which the formulation is applied externally by direct contact withthe skin surface to be treated. Conventional forms for this purposeinclude but are not limited to creams, ointments, lotions, gels, pastes,powders and the like. The term “ointments” embraces formulations(including creams) having oleaginous absorption, water-soluble, andemulsion-type bases as described in Remington's Practice of Pharmacy,11th Edition, 336 (1956).

Methods for Inhibiting Apoptosis

The present invention provides methods for the inhibition of programmedcell death, or apoptosis, by inhibition of members of the ICE/CED-3family. The invention provides new uses for not only inhibitors ofICE/CED-3 enzymatic activity, but also any method which specificallyprevents the expression of ICE/CED-3 family encoding genes. Thus,antisense RNA or DNA comprised of nucleotide sequences complementary toICE/CED-3 family member genes and capable of inhibiting thetranscription or translation of the relevant proteins, expression ofdominant negative forms of the ICE/CED-3 proteases (e.g., mutantsengineered to replace the active site cysteine with another amino acid,like serine or alanine), or antibodies which bind to ICE/CED-3 familypolypeptides, are within the scope of the invention, as are smallmolecule inhibitors, including peptides and especially the compoundspresented herein.

In a first aspect, the invention provides a method for expandinghematopoietic and blood cell populations or prolonging survival of suchpopulations, including contacting the cells with an effective amount ofa reagent which suppresses the activity of one or more ICE/CED-3 familymembers, inhibiting the programmed cell death of immature precursorsand/or mature cells, thereby expanding the cell population and/orenhancing the survival of the cell population. The term “expansion” or“expanding” as used herein means increasing the number of cells of apre-existing cell population. The term “survival” refers to maintainingviability of cells, typically ex vivo, however the term is meant toinclude in vivo as well. Survival may be from a few hours to severaldays or longer.

The method includes contacting the desired cells with an inhibitingeffective amount of a reagent which suppresses ICE/CED-3 activity. Theterm “contacting” as used herein means exposing the cells to theICE/CED-3 family inhibitor(s) such that the inhibitor(s) can effectivelyinhibit ICE/CED-3 activity thereby inhibiting apoptosis in the cells andallowing the cells to proliferate and accumulate. The term “inhibitingeffective amount” means that amount of ICE/CED-3 inhibitor thateffectively blocks ICE/CED-3 enzymatic activity in intact target cells.It will be apparent that one or more ICE/CED-3 family inhibitors can beused simultaneously in the method of the invention. In a preferredembodiment, contacting may be in vivo dosing of ICE/CED-3 inhibitor(s)to a subject in need of expansion of a cell population, such as asubject recently having undergone chemotherapy. Examples of suchreagents are commonly known in the art, including Cbz-VaLAlaAsp-CH₂F,Cbz-ValAlaAsp-CH₂OCO (2,6-diCl-C₆H₄), Cbz-ValAlaAsp-CH₂F, methyl ester,Ac-AspValAlaAsp-CH₂F. Exemplary compounds include Formula 1 and Formula3 as described supra.

Detection of ICE/CED-3 activity is by standard methods, such as anenzymatic assay to measure the fluorescence generated by enzymaticcleavage of aminomethylcoumarin (AMC) conjugated to a relevant peptide(e.g., Ac-DEVD-amc). Such assays are standard in the art (Armstrong etal., J. Biol. Chem. 271:16850, 1996); Femandes-Alnemri, et al., CancerRes., 55:6045, 1995). In addition, the inhibition of ICE activity can bemeasured by a bioassay for IL-1β. ICE/CED-3 activity is preferablysuppressed by the ICE/CED-3 family inhibitor(s) by at least about 75%,and preferably by about 90%.

The “cells” or “cell population” include precursor cells (e.g.,pluripotent stem cells) and/or differentiated, mature cells. Examples ofcells expanded and/or whose survival is enhanced by the method of theinvention include but are not limited to granulocytes (e.g.,neutrophils), monocytes, erythrocytes, lymphocytes and platelets.

Success of hematopoiesis can also be detected by measuring hematocrit,white blood cell count, incorporation of tritiated thymidine into bonemarrow DNA, spleen weight, number of burst-forming units-erythroid ornumber of colony-forming units (erythroid, granulocyte/macrophage andmegakaryocyte forming lineages) from spleen or bone marrow for example.

Hematopoietic disorders can occur as a result of primary disease or as aconsequence of therapy for another disease. For example, a serious sideeffect of radiation or chemotherapy is myelosuppression. Pancytopeniacan be observed following bone marrow transplantation and profoundleukopenia is often observed in AIDS. Patients with chronic renalfailure undergoing dialysis often have anemia. AIDS patients treatedwith AZT, cancer patients treated with platinum, and anemic patientswith rheumatoid arthritis often require transfusions because of anemiaand leukopenia. Various hematopoietic growth factors, such aserythropoietin, granulocyte or granulocyte-macrophage colony stimulatingfactors, or cytokines such as interleukin-1 are known to stimulatehematopoiesis and have been used to treat these conditions. However,treatment times are generally on the order of weeks, and especially forthe case of neutropenias, patients are at risk for opportunisticinfections. These therapies are usually quite expensive, as they arerecombinantly produced proteins. Thus, it would be advantageous to beable to administer a small molecule, e.g., ICE/CED-3 inhibitor, eitheralone or in combination with a growth factor to speed hematopoieticrecovery. Various cytokines including hematopoietic growth factors, areknown to both increase proliferation and inhibit apoptosis in targetcells (Koury and Bondurant, Science 248:378, 1990; Muta and Krantz, J.Cell Physiol. 156:264, 1993). However, those agents act upstream of theapoptotic cellular machinery, affecting that machinery only indirectly.The present invention provides methods to enhance cell expansion bydirectly inhibiting the apoptotic machinery. The combination therapycomprising administering a hematopoietic growth factor together with themethods of the present invention is a preferable embodiment.

The method of the invention is useful for restoring normal hematopoiesisin individuals in need of reconstitution of their bone marrow. Thepluripotent stem cell has the unique capacity for self-renewal and thepotential for growth and differentiation along granulocytic, monocytic,erythroid, megakaryocytic, and lymphoid lineages.

As one skilled in the art will also appreciate, stem cells can bedirected to differentiation as a lymphoid, myeloid, or erythroid-lineagepopulation by incubation with the appropriate hematopoietic growthfactor or combination of growth factors. Therefore, the method of theinvention further includes contacting the cells with a suitablehematopoietic growth factor, in addition to the ICE/CED-3 inhibitor. Asused herein, the term “a suitable hematopoietic growth factor” means oneor more hematopoietic growth factors known in the art to direct aprogenitor stem cell to the desired lymphoid, granulocytic, monocytic,megakaryocytic, myeloid, or erythroid-lineage cell population. Variousgrowth factors function both by promoting proliferation of cells and byinhibiting apoptosis; both of these functions promote the accumulationof cells. Certain hematopoietic growth factors, including G-CSF, GM-CSF,and erythropoietin are examples of such growth factors that can inhibitprogrammed cell death of their respective target cells, as well asstimulate proliferation of those cells. These factors have provenclinically useful in promoting the repopulation of granulocytes (e.g.,neutrophils), monocytes and erythrocytes in patients. G-CSF and GM-CSFare often used in patients following chemotherapy or radiation, anderythropoietin is commonly used in patients undergoing kidney dialysis.Other representative growth factors known in the art that can be usedfor these purposes are IL-1, IL-6, stem cell factor, and IL-3. Othergrowth factors for stimulating proliferation of hematopoietic lineageswill be known to those of skill in the art (see, for example, Harrison'sPrinciples of Internal Medicine, Isselbacher, et al., eds., pp1714-1717, McGraw Hill, 1994, incorporated herein by reference).

Apoptosis inhibitors, such as ICE/CED-3 inhibitors, can extend survivalof blood cell precursors in the bone marrow as well as extend survivalof the mature blood cells themselves. Regarding extension of survival ofmature blood cells, this may be particularly valuable for repopulationof granulocytes following chemotherapy and/or radiation (with or withoutbone marrow transplant). Mature granulocytes (specifically neutrophils)last for only 24 hours in the bloodstream after which they die byapoptosis. ICE/CED-3 inhibitors that block apoptosis and increaseneutrophil survival would increase the rate at which a patientnormalized his white blood cell count. Inhibition of apoptosis inprecursor cells which ultimately give rise to the mature cellpopulations would be synergistic with effects on mature cells.

The invention provides methods to preserve the viability ofneutrophils/granulocytes ex vivo for subsequent transfusion intorecipients in need of additional granulocytes. The life-span ofgranulocytes removed from donors is limited and thus it is difficult toobtain supplies of functional granulocytes for transfusion.

The disease states which may be treated or prevented by the instantcompositions include, but are not limited to, inflammatory diseases,autoimmune diseases and neurodegenerative diseases, and for inhibitingunwanted apoptosis involved in ischemic injury, such as ischemic injuryto the heart (e.g., myocardial infarction), brain (e.g., stroke), andkidney (e.g., ischemic kidney disease). As a consequence of theirability to inhibit apoptosis, the present pharmaceutical compositionsare also useful for the repopulation of hematopoietic cells of a patientfollowing chemotherapy. Methods of administering an effective amount ofthe above-described pharmaceutical compositions to mammals, alsoreferred to herein as patients, in need of such treatment (that is,those suffering from inflammatory diseases, autoimmune diseases,neurodegenerative diseases and for the repopulation of hematopoieticcells in cancer patients who have undergone chemotherapy) are anotheraspect of the instant invention. Finally, as a further consequence oftheir ability to inhibit apoptosis, the instant pharmaceuticalcompositions may be used in a method to prolong the viability of organsto be used in transplantations.

Inflammatory disease which may be treated or prevented include, forexample, septic shock, septicemia, and adult respiratory distresssyndrome. Target autoimmune diseases include, for example, rheumatoid,arthritis, systemic lupus erythematosus, scleroderma, chronicthyroiditis, Graves' disease, autoimmune gastritis, insulin-dependentdiabetes mellitus, autoimmune hemolytic anemia, autoimmune neutropenia,thrombocytopenia, chronic active hepatitis, myasthenia gravis andmultiple sclerosis. Target neurodegenerative diseases include, forexample, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson'sdisease, and primary lateral sclerosis. The pharmaceutical compositionsof this invention may also be used to promote wound healing. Targetdiseases associated with harmful, apoptosis, in other words, thoseassociated with ischemic injury, includes myocardial infarction, stroke,and ischemic kidney disease. The pharmaceutical compositions of thisinvention may also be used to treat infectious diseases, especiallythose involved with viral infections.

It is clear to those of ordinary skill in the art that the presentmethods and compositions have a variety of uses. For example, manycosmetics are known to be allergens or cause irritation to the skin.However, in combination with a reagent that suppresses the ICE/Ced-3family of proteases inflammatory responses and/or symptoms associatedwith the irritating cosmetic are substantially diminished.

Further, other inflammation inducing conditions may be treated toameliorate symptoms associated with inflammation or to diminish theexisting inflammation. Inflammation or irritation associated therewithmay be from a variety of sources either physical or chemical as notedabove, and may include: insect bites or stings, contact with aparticular type plant (e.g., poison oak, etc.), radiation (e.g., U.V.),non-infectious conjunctivitis, hemorrhoids (acute), abrasions, ingrownfinger or toenail (granulation), skin graft donor sites, vaginitis,psoriasis, herpes simplex (cold sores, aphthous ulcers), pruritisani/cruri, chemical inflammation, and the like. Moreover, suchinflammation or other activities of the ICE/ced3 familiy of proteasesmay lead to lack of elasticity or diminished skin appearance andtexture. Accordingly, the compositions and methods set forth herein,find utility not only in treating inflammatory diseases, but also for intreatment of the associated conditions and symptoms.

Inflammation is the result of extraneously induced damage to cells ortissue. Such damage may be induced by chemical and/or physicalinfluences upon the skin or mucus membranes of humans and animals.Examples of physical influences are infarction, heat, cold, radiationand electrical shock, and examples of chemical influences are contactwith acids, bases and allergens. Inflammation may be induced bymicroorganisms acting on the skin, as well as being the result ofmicroorganisms invading the human or animal body.

A variety of symptoms are associated with inflammation and include, butare not limited to one or more of the following: pain, increased surfacetemperature, swelling, itching, and reduced or ceased function.

The inflammatory responses that may be ameliorated may be on the skin ora mucus membrane of an animal and includes, but is not limited to,conditions such as inflammation around erupting wisdom teeth, followingextraction of teeth, periodontal abscesses, prosthesis induced pressuresores on the mucosa, fungal infections, for treating exposed bonesurface in alveolitis sicca dolorosa, which is a painful condition whichmay arise following extraction of teeth, chronic and acute inflammatorydiseases including, but not limited to, pancreatitis, rheumatoidarthritis, osteoarthritis, asthma, inflammatory bowel disease, psoriasisand in certain neurological disorders such as Alzheimer's disease.

Several morphological changes, including a decreased moisture content ofthe stratum corneum, coupled with reduced eccrine and sebaceous glandoutput can decrease the presence of these components which protect theskin and allow for loss of collagen, the major skin protein. Thesemorphological changes which result in a loss of integrity of the hornylayer of the skin can be caused by a variety of conditions. Among suchconditions are environmental, e.g., sun or wind exposure, trauma orwounds, e.g., cuts, burns or abrasions, exposure to chemicals such asalkaline soaps, detergents, liquid solvents, oils, preservatives, anddisease, e.g., eczema, psoriasis, seborrheic dermatitis. Accordingly,compositions and methods that suppress the protease activity of theICE/ced3 family of proteases are useful in maintaining the skin.

ICE/ced3 protease suppressors and compositions thereof are also usefulin wound repair. A wound is tissue loss or damage anywhere in the bodycaused by physical or chemical means, chronic irritation and/orinflammation of body tissue. Agents known to be useful in wound repairinclude anti-inflammatory agents and locally applied agents useful inthe production of collagen and fibrous tissue.

As those of ordinary skill in the art can readily appreciate, veterinaryuses are also within the scope of the present invention. In oneembodiment, the compositions for cosmetic, pharmaceutical ordermatological usage which comprises at least one agent that suppressesthe protease activity of a member of the ICE/ced3 family of proteases ora cosmetically, pharmaceutically or dermatologically acceptable saltthereof are contained in a cosmetically, pharmaceutically ordermatologically acceptable medium, wherein said at least one producthaving the irritant side effect is contained in an amount which wouldotherwise cause irritation in the absence of an anti-irritant effectiveamount of said at least one agent that suppresses the protease activityof a member of the ICE/ced3 family of proteases.

In certain embodiments, the product having an irritant side effect maybe from the group consisting of α-hydroxy acids, α-hydroxy acids, α-ketoacids, α-keto acids, retinoids, anthralins, anthranoids, peroxides,minoxidil, lithium salts, antimetabolites, vitamin D, hair dyes, hairtoners, antiperspirants, depilatory agents, permanent-waving agents,perfumed alcoholic solutions, depigmenting agents, surfactants andsolvents.

The reagents of the present invention are “ICE/CED-3 inhibitors” in thatthey inhibit the catalytic activity of members of the. ICE/CED-3 familyin a reversible or an irreversible manner. The term “irreversible” asused herein means the formation of a covalent bond between the ICE/CED-3family member and the inhibitor. It is possible to convert a reversibleinhibitor to an irreversible inhibitor by incorporating an irreversible“warhead” into what would otherwise be a reversible inhibitor.

The reversibility of ICE/CED-3 inhibition is generally a function of theelectronegative group in the molecule. When the electronegative group isa diazoalkyl ketone, the inhibition of ICE activity is irreversible andthe compound is an irreversible inhibitor. When the electronegativegroup is an aldehyde, the inhibition of ICE is reversible and theinhibitor is a reversible inhibitor.

A compound of the invention preferably has an aldehyde, a diazoalkylketone, a haloalkyl ketone, or acyloxymethyl ketone. As used herein inreference to an electronegative group, “alkyl” refers to linear orbranched chain radicals having 1-3 carbon atoms, which may be optionallysubstituted. Representative alkyl groups include methyl, ethyl, propyland the like. Optionally, the electronegative group is an aldehyde,fluoromethyl (CH₂F) ketone, or acyloxylmethyl ketone.

The compounds of the present invention are made by techniques generallyto methods known and readily apparent to those of skill in the art. See,e.g., Kettner, et al., Arch, Biochem, Biophys., 162:56, 1974; U.S. Pat.No. 4,582,821; U.S. Pat. No. 4,644,055; Kettner, et al. Arch, Biochem,Biophys, 165:739, 1974; Dakin and West, J. Biol. Chem., 78:91, 1928;Rasnick, D., Anal. Biochem., 149:461, 1985; Revesz, L., TetrahedronLett., 35:9693, 1994. Exemplary indolyl dipeptide and tricycliccompounds are provided herein.

Compounds having a non-fluoro, haloalkyl ketone electronegative leavinggroup are preferably synthesized in accordance with the Kettnerprocedure. An N-blocked amino acid or peptide is reacted withN-methylmorpholine and an alkyl, non-fluoro haloformate to generate apeptide-acid anhydride. The anhydride is then reacted with a diazoalkanein an inert, aprotonic solvent to form a peptide-diazomethane ketone.The diazomethane ketone is then reacted with an anhydrous solution ofHCl, HBr or HI to produce the desired N-blocked, C-terminal haloalkylketone peptide or amino acid.

Compounds having a fluoromethyl electronegative leaving group arepreferably synthesized by the Revesz procedure. An N-blocked peptide oramino acid is reacted with t-butyl (3-amino-4-hydroxy-5-fluoro)pentanoate in the presence of a standard peptide coupling agent such asdicyclohexylcarbodiimide-hydroxy-benztriazole. The resulting product isoxidized to the corresponding ketone by either Severn or Dess-Martinoxidation. Finally, deprotection of the t-butylester with trifluoraceticacid gives the corresponding carboxylic acid.

Compounds having a fluoroalkyl ketone electronegative leaving group canbe extended in the N-terminus direction by removing the N-terminalblocking group and coupling the deprotected compound with otherprotected amino acids. Bodanszky, The Practice of Peptide Synthesis,Springer-Verlag, Berlin, 1984. Alternatively, deprotected compounds areacetylated to yield compounds having an N-terminal acetyl protectinggroup. Stewart, et al., Solid Phase Peptide Synthesis, Pierce ChemicalCo., Rockford, Ill., 1984.

In another embodiment, the invention provides a method for prolongingorgan viability comprising contacting the cells of an organ fortransplantation with an inhibiting effective amount of a reagent whichsuppresses interleukin-1β-converting enzyme (ICE)/CED-3 activity,thereby prolonging the viability of the organ as compared to anuntreated organ. The term “organ” is meant to include intactmulti-cellular organs such as kidney, liver, or heart; cell suspensionsderived from multi-cellular organs (e.g., pancreatic islet cells indopaminergic neurons); as well as suspensions of blood cells orhematopoietic precursor cells. Preferably, an organ is treated ex vivoin order to preserve the organ for transplantation. The term“prolonging” means that an organ for transplantation is preserved bytreatment using the method of the invention as compared to a similarorgan that has not been treated with an ICE/CED-3 inhibitor. While notwanting to be bound by a particular theory, it is believed thatcontacting the cells of an organ for transplantation with an ICE/CED-3inhibitor, inhibits programmed cell death, thereby preserving the organand prolonging viability.

The method of the invention includes treatment of the recipient priorand subsequent to transplantation with genetically altered or ICE/CED-3inhibitor-bathed donor organs to inhibit apoptosis of donor organ cells,and optionally, an immunosuppressive agent.

The present invention includes the ex vivo expansion or survival ofhematopoietic cells which is useful for a variety of clinical usesincluding gene therapy, augmentation of bone marrow transplantation, andthe replacement of bone marrow transplantation. Therefore, the use of aninhibitor of the apoptosis machinery, such as an ICE/CED-3 familyinhibitor, to promote the ex vivo expansion and/or survival ofpopulations of granulocytes, erythrocytes, lymphocytes and or plateletconstitutes a useful method. For example, one can add the ICE/CED-3inhibitor(s) to a tissue culture of cells isolated from a subject, or bytransfecting the cells with an expression vector containing an operableICE/CED-3 inhibitor-encoding polynucleotide, e.g., antisense, to suchcells.

The method of the invention has therapeutic utility. For instance, asuspension of pluripotent hematopoietic stem cells (HSC) obtained bystandard methods in the art, can be treated by the method of theinvention and can be used for performing hematopoietic reconstitution ofa recipient. As an ex vivo treatment for example, enriched pluripotentstem cells derived from the recipient (autologous reconstitution) orderived from an individual other than the recipient (non-autologousreconstitution) can be expanded and used in the treatment or preventionof various diseases or disorders such as anemias, malignancies,autoimmune disorders, and various immune dysfunctions and deficiencies,as well as recipients whose hematopoietic cellular repertoire has beendepleted, such as recipients treated with various chemotherapeutic orradiologic agents, or recipients with AIDS. Other therapeutic uses ofthe compositions of the invention are well known to those of skill inthe art. Treatment of the transplanted cells can be both ex vivo, andfollowing transplantation, in vivo.

An example of the way in which the invention may be applied tonon-autologous donor organs for human recipients is as follows:beginning one week prior to the transplantation, the recipient may bedosed with cyclophosphamide to reduce the potential for evokedimmunological responses. An immunosuppressive dose of cyclosporine orFK506 may be started shortly (1-3 days) before transplantation toenhance graft acceptance. Immediately prior to transplantation, thedonor may be dosed with an ICE/CED-3 inhibitor, prior to organ removal.Upon removal prior to transplantation, the donor organ is flushed with asolution containing at least one ICE/CED-3 inhibitor, e.g., a peptidefluoroalkyl ketone. Following transplantation by standard surgicaltechniques, the patient is typically maintained on routineimmunosuppression using cyclosporine or FK506, cyclophosphamide, andsteroids and optionally, the ICE/CED-3 inhibitor(s). Based on clinicalsigns and symptoms related to immune responsiveness, various of theimmunosuppressants are reduced in dosage.

An immunosuppressive agent used during transplantation is an agent suchas Cyclosporine A (CsA), however other agents which cause immunesuppression, such as rapamycin, desoxyspergualine, and FK506 orfunctional equivalents of these compounds, may also be utilized. CsA ispreferably administered by injection at an immunosuppressive dose. Theduration of CsA treatment may range from about 2 to about 20 days.

For enhancing organ survival prior to transplantation, the ICE/CED-3family inhibitor may be administered by addition to the fluid used toperfuse the excised organ. If the ICE/CED-3 inhibitor is also to beadministered to the donor prior to excision of the organ, the ICE/CED-3inhibitor(s) are administered by any suitable means, includingparenteral, subcutaneous, intrapulmonary, and intranasal administration.Parenteral infusions include intramuscular, intravenous, intraarterial,or intraperitoneal administration.

It is understood that any organ from any species can be transplanted.The method of the invention is useful for preserving organs to be usedfor same species transplants such as human recipients and other humandonors (allografts and autologous grafts) or to human recipients fromother species such as sheep, pigs, or non-human primates (xenografts),for example. Such tissues for transplant include, but are not limitedto, heart, liver, kidney, lung, pancreas, pancreatic islets, braintissue, cornea, bone, intestine, skin, and hematopoietic cells. Thehuman is the preferred recipient.

In order to determine the amount of ICE/CED-3 inhibitor to administer tothe donor, e.g., pig, and the amount of ICE/CED-3 inhibitor with whichto treat the organ to be transplanted, a compound is administered sothat its concentration in the vasculature is at least as high as thatnecessary for inhibition of apoptosis in a cell culture model. A varietyof cell culture models are known in the art (e.g., Armstrong et al.,supra; Schlegel, et al., supra; Boudreau, et al., supra). The donororgan, and optionally the donor, is then dosed with an ICE inhibitor inorder to reduce apoptosis to as much as less than about 10% of thenormal apoptosis observed in the organ from the donor. Therefore, asused herein, the term “apoptosis-inhibiting” amount refers to thatamount of ICE inhibitor that inhibits ICE activity and/or apoptosis byabout 75%, and preferably by about 90%.

Method for Enhancing Bioproduction

In yet another embodiment, the invention provides a method forincreasing survival of cells cultured in vitro for utilities other thantransplantation. For example, inhibition of programmed cell death is ofuse in enhancing bioproduction processes. For example, in production ofrecombinant proteins, yields may be limited by apoptotic cell death ofcultured host cells. Therefore, treatment of host cells with ICE/CED-3inhibitors, or using host cells which express antisense RNA or DNAsequences complementary to ICE/CED-3 and capable of inhibiting thetranscription or translation of ICE/CED-3 family members or that expressgenes encoding ICE/CED-3 family inhibitors, would enhance bioproductionby allowing cells to survive longer and produce and/or secrete a desiredproduct longer, thus resulting in a greater yield of product. Theability to prevent programmed cell death may allow cells to liveindependent of normally required growth factors, reducing the cost ofmedia supplements.

In order to demonstrate utility in bioproduction, a cell line thatnaturally produces a useful product (e.g., a cytokine) or a cell linegenetically engineered to express a useful product (e.g., COS or CHOcells stably expressing recombinant human erythropoieten, growth hormoneor G-CSF) is contacted with the ICE/CED-3 inhibitor during fermentation.Effectiveness of the ICE/CED-3 inhibitor on bioproduction can bemeasured in several ways: 1) determining the percentage of apoptoticcells in the culture at different time points; 2) determining the usefullifespan of the culture with regard to production of the desiredproduct; 3) measuring the yield of product per gram of cells or pervolume of culture; 4) measuring final purity of the product.

Methods for increasing the efficiency or overall productivity ofbioproduction are useful because they reduce costs of producing anatural or recombinant product. Mammalian cell fermentation is limitedby decreasing cell viability with time. Cell death during fermentationhas been shown to be apoptotic, thus inhibitors of apoptosis willincrease cell viability during bioproduction. Growth media used forbioproduction are often serum-free supplemented with growth factors toenhance cell viability. Supplementation with ICE/CED-3 inhibitors of thepresent invention is designed to replace or augment such additives.

Pharmaceutical, Cosmetic or Dermatologic Compositions

The compositions of the invention may be in all the pharmaceutical formsnormally used, depending on whether the composition must be ingested,injected or applied to the skin or the mucous membranes.

For topical application, the composition must in particular take theform of an aqueous or oily solution or a dispersion of the lotion orserum type, emulsions of liquid or semi-liquid consistency of the milktype, obtained by dispersion of a fatty phase in an aqueous phase (O/W)or conversely (W/O), or suspensions or emulsions of smooth consistencyof the aqueous or anhydrous cream or gel type, or alternativelymicrocapsules or microparticles, microemulsion, or vesicle dispersionsof ionic and/or nonionic type. These compositions are prepared accordingto the usual methods.

They may also be used for the hair in the form of aqueous, alcoholic oraqueous-alcoholic solutions, or in the form of creams, gels, emulsionsor mousses or alternatively in the form of aerosol compositions alsocontaining a propellant under pressure.

For injection, the composition may be in the form of an aqueous or oilylotion or in the form of serum. For the eyes, it may be in the form ofdrops, and for ingestion it may be in the form of capsules, granules,syrups or tablets.

The amounts of the different constituents of the compositions accordingto the invention are those traditionally used in the fields in question.

The compositions for cosmetic or dermatologic use constitute, inparticular, cleansing, protective, treatment or skin care creams for theface, hands, feet, major anatomical folds or the body (for example daycreams, night creams, make-up removal creams, foundation creams,sun-protection creams), fluid foundations, make-up removal milks,protective or skin care body milks, after-sun milks, skin care lotions,gels or foams, such as cleansing or disinfecting lotions, bathcompositions, deodorant compositions, aftershave gels or lotions,compositions or compositions for treating certain skin disorders such asthose mentioned above.

The compositions according to the invention may also consist of solidpreparations constituting cleansing bars or soaps.

The compositions may also be packaged in the form of an aerosolcomposition also containing a propellant under pressure.

The compound of the invention may also be incorporated into varioushaircare compositions, and in particular shampoos, hairsetting lotions,treating lotions, styling creams or gels, dye compositions (inparticular oxidation dyes) optionally in the form of colouring shampoos,restructuring lotions for the hair, permanent-wave compositions (inparticular compositions for the first stage of a permanent-wavingoperation), lotions or gels for combating hair loss, antiparasiticshampoos, and the like.

The compositions of the invention may also be for dentibuccal use, forexample a toothpaste or a mouthwash. In this case, the compositions cancontain standard adjuvants and additives for compositions for buccaluse, and in particular surfactants, thickening agents, humectant agents,polishing agents such as silica, various active ingredients such asfluorides, especially sodium fluoride, and, where appropriate,sweetening agents such as saccharin sodium.

When the composition of the invention is an emulsion, the proportion ofthe fatty phase may range from 5% to 80% by weight, and preferably from5% to 50% by weight, relative to the total weight of the composition.The oils, the waxes, the emulsifiers and the coemulsifiers used in thecomposition in emulsion form are chosen from those used conventionallyin the cosmetics field. The emulsifier and the coemulsifier are presentin the composition at a proportion ranging from 0.3% to 30% by weight,and preferably of from 0.5 to 20% by weight, relative to the totalweight of the composition. The emulsion may also contain lipid vesicles.

When the composition of the invention is an oily solution or gel, thefatty phase may represent more than 90% of the total weight of thecomposition.

In a known manner, the cosmetic compositions of the invention may alsocontain adjuvants which are common in the cosmetics field, such ashydrophilic or lipophilic gelling agents, hydrophilic or lipophilicadditives, preserving agents, antioxidants, solvents, fragrances,fillers, screening agents, odour absorbers and dyestuffs. The amounts ofthese various adjuvants are those used conventionally in the cosmeticsfield and, for example, from 0.01% to 10% of the total weight of thecomposition. Depending on their nature, these adjuvants may beintroduced into the fatty phase, into the aqueous phase and/or into thelipid spherules.

As oils or waxes which may be used in the invention, there may bementioned mineral oils (liquid petrolatum), plant oils (liquid fractionof karite butter, sunflower oil), animal oils (perhydrosqualene),synthetic oils (purcellin oil), silicone oils or waxes (cyclomethicone)and fluoro oils (perfluoropolyethers), beeswaxes, carnauba wax orparaffin wax. Fatty alcohols and fatty acids (stearic acid) may be addedto these oils.

As emulsifying agents which may be used in the invention, there may bementioned for example glyceryl stearate, polysorbate 60 and thePEG-6/PEG-32/glycol stearate mixture sold under the name TEFOSE® 63 bythe company Gattefosse.

As solvents which may be used in the invention, there may be mentionedlower alcohols, in particular ethanol and isopropanol, and propyleneglycol.

Hydrophilic gelling agents which may be useful are carboxyvinyl polymers(carbomer), acrylic copolymers such as acrylate/alkylacrylatecopolymers, polyacrylamides, polysaccharides such as hydroxypropylcellulose, natural gums and clays, and, as lipophilic gelling agents,there may be mentioned modified clays such as bentones, fatty acid metalsalts such as aluminium stearates, and hydrophobic silica, ethylcellulose and polyethylene.

As hydrophilic active agents, proteins or protein hydfolysotes, aminoacids, polyols, urea, allantoin, sugars and sugar derivatives,water-soluble vitamins, starch and plant extracts, in particular thoseof aloe vera may be used.

The compositions of the invention may contain other hydrophilic activeagents such as proteins or protein hydrolysates, amino acids, polyols,urea, allantoin, sugars and sugar derivatives, water-soluble vitamins,plant extracts and hydroxy acids.

Lipophilic active agents which may be used are retinol (vitamin A) andderivatives thereof, tocopherol (vitamin E) and derivatives thereof,essential fatty acids, ceramides, essential oils, and salicylic acid andderivatives thereof.

It is possible, inter alia, to combine the agents that suppress theprotease activity of a member of the ICE/ced3 family of proteases, whichis optionally salified, with other active agents intended in particularfor the prevention and/or treatment of skin complaints. These activeagents include, but are not limited to:

agents which modify cutaneous differentiation and/or proliferationand/or pigmentation such as retinoic acid and isomers thereof, retinoland esters thereof, vitamin D and derivatives thereof, oestrogens suchas oestradiol, kojic acid or hydroquinone;

antibacterial agents such as clindamycin phosphate, erythromycin orantibiotics from the tetracycline family;

antiparasitic agents, in particular metronidazole, crotamiton orpyrethroids;

antifungal agents, in particular compounds belonging to the imidazolefamily such as econazole, ketoconazole or miconazole or salts thereof,polyene compounds such as amphotericin B, compounds from the allylaminefamily such as terbinafine, or alternatively octopirox;

steroidal anti-inflamatory agents such as hydrocortisone, betamethasonevalerate or clobetasol propionate, or nonsteroidal anti-inflaoatoryagents such as ibuporofen and salts thereof, diclofenac and saltsthereof, acetylsalicylic acid, acetaminophen or glycyrrhetinic acid;

anaesthetics such as lidocaine hydrochloride and derivatives thereof;

antipruriginous agents such as thenaldine, trimeprazine orcyproheptadine;

antiviral agents such as acyclovir;

keratolytic agents such as alpha- and beta-hydroxycarboxylic acids orbeta-ketocarboxylic acids, the salts, amides or esters thereof and moreparticularly hydroxy acids such as glycolic acid, lactic acid, salicylicacid, citric acid and fruit acids in general, and 5-n-octanoylsalicylicacid;

anti-free-radical agents such as alpha-tocopherol or esters thereof,superoxide dismutases, certain metal chelating agents or ascorbic acidand esters thereof;

anti-seborrhoeic agents such as progesterone;

anti-dandruff agents such as octopirox or zinc pyrithione;

anti-acne agents such as retinoic acid or benzoyl is peroxide.

Agents that inhibit the protease activity of a member of the ICE/ced3family of proteases may be combined with products having an irritantside-effect, and in particular active agents commonly used in thecosmetics, pharmaceutical or dermatological field. The presence of anagent that inhibits the protease activity of a member of the ICE/ced3family of proteases may be in a cosmetic, pharmaceutical ordermatological composition containing a product or even an active agenthaving an irritant effect enables this irritant effect to be greatlyattenuated or even eliminated. Further, those of ordinary skill in theart would appreciate that in any of the aforementioned compositions thatother anti-inflammatory agents may be combined therewith.

In particular, agents that suppress the activity of an ICE/ced3 proteasemake it possible, in particular, to increase the amount of cosmetic,pharmaceutical or dermatological active agent relative to the amountnormally used, with a view to improved efficacy.

Thus, in one embodiment of the invention is also a compositioncontaining, in a cosmetically, pharmaceutically or dermatologicallyacceptable medium, at least one product having an irritant side-effect,characterized in that it contains at least one agent that suppresses theactivity of a protease of the ICE/ced3 family and combinations thereof.

The agents are also useful for the treatment of inflammation in asubject, and for treatment of other inflammation-associated disorders,for example, as an analgesic in the treatment of pain and headaches, oras an antipyretic for the treatment of fever. For example, thecompositions would be useful to treat arthritis, including but notlimited to rheumatoid arthritis, spondyloarthopathies, gouty arthritis,systemic lupus erythematosus, osteoarthritis and juvenile arthritis.Such compositions would be useful in the treatment of asthma,bronchitis, menstrual cramps, tendinitis, bursitis, and skin relatedconditions such as psoriasis, eczema, bums and dermatitis. Thecompositions are also useful to treat gastrointestinal conditions suchas inflammatory bowel syndrome, Crohn's disease, gastritis, irritablebowel syndrome and ulcerative colitis. The compositions are also usefulin treating inflammation in such diseases as vascular diseases, migraineheadaches, periarteritis nodosa, thyroiditis, aplastic anemia, Hodgkin'sdisease, sclerodoma, rheumatic fever, type I diabetes, myastheniagravis, sarcoidosis, nephrotic syndrome, Behcet's syndrome,polymyositis, hypersensitivity, conjunctivitis, gingivitis, swellingoccurring after injury, myocardial ischemia, and the like.

Pharmaceutical compositions of this invention comprise any of thecompounds of the present invention, and pharmaceutically acceptablesalts thereof, with any pharmaceutically acceptable carrier, adjuvant orvehicle (hereinafter collectively referred to as“pharmaceutically-acceptable carriers”). Pharmaceutically acceptablecarriers, adjuvants and vehicles that may be used in the pharmaceuticalcompositions of this invention include, but are not limited to, alumina,aluminum stearate, lecithin, serum proteins, such as human serumalbumin; buffer substances such as the various phosphates, glycine,sorbic acid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids; water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, and zinc salts; colloidal silica, magnesiumtrisilicate, polyvinyl pyrrolidone, cellulose-based substances,polyethylene glycol, sodium carboxymethylcellulose, polyarylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat, and the like.

The pharmaceutical compositions of this invention may be administeredorally, parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or by an implanted reservoir. Oral and parenteraladministration are preferred. The term “parenteral” as used hereinincludes subcutaneous, intracutaneous, intravenous, intramuscular,intra-articular, intrasynovial, intrasternal, intrathecal, intralesionaland intracranial injection or infusion techniques.

The pharmaceutical compositions may be in the form of a sterileinjectable preparation, for example, as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according totechniques known in the art using suitable dispersing or wetting agents(such as, for example, Tween 80) and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are mannitol, water, Ringer'ssolution and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose, any bland fixed oil may be employed includingsynthetic mono- or diglycerides. Fatty acids, such as oleic acid and itsglyceride derivatives are useful in the preparation of injectables, asare natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, and aqueous suspensions and solutions. Inthe case of tablets for oral use, carrier which are commonly usedinclude lactose and corn starch. Lubricating agents, such as magnesiumstearate, are also typically added. For oral administration in capsuleform useful diluents include lactose and dried corn starch. When aqueoussuspensions are administered orally, the active ingredient is combinedwith emulsifying and suspending agents. If desired, certain sweeteningand/or flavoring and/or coloring agents may be added.

The pharmaceutical compositions of this invention may also beadministered in the form of suppositories for rectal administration.These compositions can be prepared by mixing a compound of thisinvention with a suitable non-irritating excipient which is solid atroom temperature but liquid at the rectal temperature. Such materialsinclude, but are not limited to, cocoa butter, beeswax and polyethyleneglycols.

Topical administration of the pharmaceutical compositions of thisinvention is especially useful when the desired treatment involves areasor organs readily accessible to topical application. For applicationtopically to the skin, the pharmaceutical composition should beformulated with a suitable ointment containing the active componentssuspended or dissolved in a carrier. Carriers for topical administrationof the compounds of this invention include, but are not limited to,mineral oil, liquid petroleum, white petroleum, propylene glycol,polyoxyethylene, polyoxypropylene compound, emulsifying wax and water.Alternatively, the pharmaceutical composition can be formulated with asuitable lotion or cream containing the active compound suspended ordissolved in a carrier. Suitable carriers include, but are not limitedto, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esterswax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. Thepharmaceutical compositions of this invention may also be topicallyapplied to the lower intestinal tract by rectal suppository formulationor in a suitable enema formulation. Topically applied transdermalpatches are also included in this invention.

The pharmaceutical compositions of this invention may be administered bynasal aerosol or inhalation. Such compositions are prepared according totechniques well-known in the art of pharmaceutical formulation and maybe prepared as solutions in saline, employing benzyl alcohol or othersuitable preservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other solubilizing or dispersing agents known inthe art.

The compounds of this invention may be used in combination with eitherconventional anti-inflammatory agents or with matrix metalloproteaseinhibitors, lipoxygenase inhibitors and antagonists of cytokines otherthan IL-1β.

When the compounds of this invention are administered in combinationtherapies with other agents, they may be administered sequentially orconcurrently to the patient. Alternatively, pharmaceutical compositionsaccording to this invention may be comprised of a combination of acompound of Formula 1 or 3 or other ICE inhibitors as described herein,and another therapeutic or prophylactic agent mentioned above.

The term effective amount” refers to dosage levels of the order of fromabout 0.05 milligrams to about 140 milligrams per kilogram of bodyweight per day for use in the treatment of the above-indicatedconditions (typically about 2.5 milligrams to about 7 grams per patientper day). For example, inflammation may be effectively treated by theadministration of from about 0.01 to 50 milligrams of the compound perkilogram of body weight per day (about 0.5 milligrams to about 3.5 gramsper patient per day). The amount of the compounds of Formula 1, 3, orother ICE/ced-3 inhibitors that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. For example, aformulation intended for the oral administration of humans may containfrom 0.5 milligrams to 5 grams of a compound of Formula 1 combined withan appropriate and convenient amount of a pharmaceutically-acceptablecarrier which may vary from about 5 to about 95 percent of the totalcomposition. Dosage unit forms will generally contain between from about1 milligram to about 500 milligrams of an active compound of Formula 1,3, or other ICE/ced-3 inhibitors.

It will be understood, however, that the specific “effective amount” forany particular patient will depend upon a variety of factors includingthe activity of the specific compound employed, the age, body weight,general health, sex, diet, time of administration, route ofadministration, rate of excretion, drug combination and the severity ofthe particular disease undergoing prevention or therapy.

Although this invention focuses on the use of the compounds disclosedherein for inhibiting ICE/CED-3 proteases and for preventing apoptosis,the compounds of this invention can also be used as inhibitory agentsfor other cysteine proteases.

The compounds of this invention are also useful as commercial reagentswhich effectively bind to the ICE/ced-3 family of cysteine protease orother cysteine proteases. As commercial reagents, the compounds of thisinvention, and their derivatives, may be used to block proteolysis of atarget peptide or may be derivatized to bind to a stable resin as atethered substrate for affinity chromatography applications. These andother uses which characterize commercial cystine protease inhibitorswill be evident to those of ordinary skill in the art.

The following examples are intended to illustrate, but not limit theinvention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may alternatively be used.

In the following Examples, proton NMR spectra were obtained at 300 MHZ;chemical shifts are quoted downfield from internal tetramethylsilane.

EXAMPLES Example 1 Assays for Inhibition of ICE/CED-3 Protease FamilyActivity

A. Determination of IC₅₀ Values

Fluorescence enzyme assays detecting the activity of the compounds ofFormula 1 utilizing the recombinant ICE and cpp32 enzymes were performedessentially according to Thomberry et al., Nature 356:768:774 (1992);and Nicholson et al., Nature 376:37-43 (1995), respectively, (hereinincorporated by reference) in 96 well microtiter plates. The substratefor these assays was Acetyl-Tyr-Val-Ala-Asp-amino-4-methylcoumarin (AMC)for the ICE assay and Acetyl-Asp-Glu-Val-Asp-amino-4-methylcoumarin forthe cpp32 and Mch5 assay. Enzyme reactions were run in ICE buffer (25 mMHEPES, 1 mM EDTA, 0.1% CHAPS, 10% sucrose, pH 7.5) containing 2 mM DTTat room temperature in duplicate. The assays were performed by mixingthe following components:

50 μl of either ICE, Mch2, Mch5 or cpp32 (18.8, 38, 8.1 and 0.153 nMconcentrations, respectively) or Mch3 (1 unit) enzyme in ICE buffercontaining either 8.0 (ICE, Mch2, Mch3, cpp32) or 20 (Mch5) mM DTT;

50 μl of either the compound of Formula 1 or ICE buffer (control); and

100 μl of 20 μM substrate.

The enzyme and the compound of Formula 1 to be assayed were preincubatedin the microtitre plate wells for 30 minutes at room temperature priorto the addition of substrate to initiate the reaction. Fluorescent AMCproduct formation was monitored for one hour at room temperature bymeasuring the fluorescence emission at 460 nm using an excitationwavelength of 360 nm. The fluorescence change in duplicate (control)wells were averaged and the mean values were plotted as a function ofinhibitor concentration to determine the inhibitor concentrationproducing 50% inhibition (IC₅₀). The results are set forth in Tables 1and 4 (FIGS. 1 and 4).

The reference compound for this assay was Cbz-VaLAlaAsp-H and the valuesare denoted in Tables 1 and 4 as “Reference”.

B. Determination of the Dissociation Constant K_(i) and IrreversibleRate Constant k_(S) for Irreversible Inhibitors

For the irreversible inhibition of a ICE/ced-3 Family Protease enzymewith a competitive irreversible inhibitor; using the model representedby the following formulas:${{E + 1}\overset{K_{i}}{\rightleftharpoons}{EI}}\overset{k_{3}}{\rightarrow}{E - 1}$${{E + S}\overset{K_{s}}{\rightleftharpoons}{ES}}\overset{k_{s}}{\rightarrow}{E + {P.}}$

The product formation at time t may be expressed as: $\begin{matrix}{\lbrack P\rbrack_{t} = {\lbrack E\rbrack^{T}\left( \frac{\lbrack S\rbrack K_{i}}{\lbrack I\rbrack K_{s}} \right){\left( \frac{k_{s}}{k_{3}} \right)\left\lbrack {1 - ^{{- k_{3}}{t{({1 + {\frac{K_{i}}{\lbrack I\rbrack}{({1 + \frac{\lbrack S\rbrack}{K_{s}}})}}})}}}} \right\rbrack}}} & {{Equation}\quad 1}\end{matrix}$

where E, I, EI, and E-I denote the active enzyme, inhibitor,non-covalent enzyme-inhibitor complex and covalent enzyme-inhibitoradduct, respectively. The K_(i) value is the overall dissociationconstant of reversible binding steps, and k₃ is the irreversible rateconstant. The [S] and K_(S) values are the substrate concentration andthe dissociation constant of the substrate bound to the enzyme,respectively.

The above equations were used to determine the K_(i) and k₃ values of agiven inhibitor bound to a ICE/ced-3 family protease. Thus, a continuousassay was run for sixty minutes at various concentrations of theinhibitor and the substrate. The assay was formulated essentially thesame as described above for generating the data in Table 1, except thatthe reaction was initiated by adding the enzyme to thesubstrate-inhibitor mixture. The Ki and k₃ values were obtained bysimulating the product AMC formation as a function of time according toEquation I. The results of this second assay are set forth below inTables 2, 3 and 5 (FIGS. 2, 3 and 5).

The reference compound for this assay was Cbz-ValAlaAsp-CH₂ F and thevalues are denoted in Tables 2, 3 and 5 as “Reference”.

Example 2(3S)-3-[(1-Methylindole-2-carbonyl)alaninyl]amino-4-oxobutanoic Acid,t-Butyl Ester Semicarbazone

1-Methylindole-2-carboxylic acid (107 mg, 0.6 mmol) and(3S)-3-(alaninyl)-amino-4-oxobutanoic acid, t-butyl ester semicarbazone(188 mg, 96%, 0.6 mmol) were dissolved in DME (2 mL) then both1-hydroxybenzotriazole-hydrate (96 mg, 0.63 mmol) and1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDAC) (161mg, 0.84 mmol) was added to the resultant mixture under a nitrogenatmosphere at 0° C. Stirring was continued for 1 hour at 0° C. and anadditional 20 hours at room temperature. The reaction mixture wasdiluted with ethyl acetate, washed successively with saturated aqueoussodium bicarbonate solution and brine, dried over sodium sulfate andconcentrated to give a yellow solid. Trituration of the solid with etherafforded the title product as a slightly yellow powder (213 mg, 77%).TLC: (methanol/methylene chloride: 1/9, silica gel): R_(f)=0.47; ¹H NMR(CDCl₃+CD₃ OD): δ 7.96 (d, J=8.0, 1H), 7.57-7.67 (m 2H), 7.31-7.42 (m,2H), 7.13-7.19 (m, 2H), 7.06 (s, 1H), 4.91 (m, 1H), 4.65 (q, J=7.1, 1H),4.01 (s, 3H1), 2.59-2.78 (m, J=5.6, 15.7, 2H), 1.49 (d, J=7.1, 3H), 1.39(s, 9H).

Example 3(3S)-3-[(1-Methylindole-2-carbonyl)alaninyl]amino-4-oxobutanoic acid,Semicarbazone

(3S)-3-[(1-Methylindole-2-carbonyl)alaninyl] amino-4-oxobutanoic acid,t-butyl ester semicarbazone (127 mg, 0.28 mmol) was suspended in anisole(0.2 mL) and methylene chloride (2 mL) and the suspension was treatedwith trifluroacetic acid (TFA) 1 mL). The resulting solution was stirredfor 2 hours under a nitrogen atmosphere at room temperature. Thereaction mixture was then concentrated and chased with methylenechloride to give a purple foam. Trituration of the foam with ether gavethe title product as a purple powder (108 mg, 97%). TLC: (methylenechloride:methanol:acetic acid, 20:1:1, silica gel): R_(f)=0.27; ¹H NMR(CD₃ OD): δ 7.62 (d, J=8.0, 1H), 7.44 (d, J=8.2, 1H), 7.24-7.32 (m, 2H),7.07-7.13 (m, 2H), 4.91 (m, 1H), 4.56 (q, J=7.1, 1H), 3.98 (s, 3H), 2.78(d, J=6.5, 2H), 1.49 (d, J=7.3, 3H).

Example 4(3S)-3-[(1-Methylindole-2-carbonyl)alaninyl]amino-4-oxobutanoic Acid

(3S)-3-[(1-Methylindole-2-carbonyl)alaninyl] amino-4-oxobutanoic acid,semicarbazone (87 mg, 0.22 mmol) was dissolved in methanol (3 mL),formaldehyde (1 mL, 37% wt. aq) and acetic acid (1 mL) and the resultantmixture was stirred for 4 hours under a nitrogen atmosphere at roomtemperature. The reaction mixture was diluted with water and extractedtwice with ethyl acetate. The ethyl acetate solution was washed withbrine, dried over sodium sulfate and concentrated to give a glassymaterial which was triturated with ether to afford the title product asa gray powder (24 mg, 32%). TLC: (methylene chloride:methanol:aceticacid, 20:1:1, silica gel): R_(f)=0.44; ¹H NMR (CD₃ OD): Δ 7.62 (d,J=8.0, 1H), 7.44 (dd, J=0.8, 8.4, 1H), 7.26-7.32 (m, 1H), 7.08-7.13 (m,2H), 4.63-4.53 (m, 2H), 4.31 (m, 1H), 3.99 (s, 3H), 2.48-2.73 (m, 2H),1.46 (7.1, 3H).

Example 5(3S)-3-[(1-Methylindole-2-carbonyl)prolinyl]amino-4-oxobutanoic Acid,t-Butyl Ester Semicarbazone

1-Methylindole-2-carboxylic acid (102 mg, 0.58 mmol) and(3S)-3-(prolinyl)amino-4-oxobutanoic acid, t-butyl ester semicarbazone(189 mg, 0.58 mmol) were dissolved in methylene chloride (2 mL) and DMF(1 mL) and then both 4-dimethylamino pyridine (DMAP) (71 mg, 0.58 mmol)and EDAC (155 mg, 0.81 mmol) were added to the mixture under a nitrogenatmosphere at 0° C. Stirring was continued for 1 hour at 0° C. and anadditional 2 hours at room temperature. The reaction mixture waspartitioned between ethyl acetate and 5% KHSO₄ solution. The ethylacetate solution was washed with saturated sodium bicarbonate solutionand brine, dried over sodium sulfate and concentrated to give 153 mg ofbrown foam. The foam was purified by flash chromatograph on silica gelusing 2% methanol-methylene chloride as the eluant to give the titleproduct as a light brown foam (50 mg). TLC: (methanol/methylenechloride: 5/95, silica gel): R_(f)=0.27; ¹H NMR (CDCl₃+CD₃ OD): δ 8.87(bs, 1H), 7.63 (d, J=7.7, 1H), 7.38-7.50 (m, 2H), 7.17-7.13 (m, 1H),6.85 (bs, 1H), 4.90-4.81 (m, 2H), 3.92-3.74 (m, 5H), 2.78-1.93 (m, 6H1),1.37 (s, 9H).

Example 6(3S)-3-[(1-Methylindole-2-carbonyl)prolinyl]amino-4-oxobutanoic Acid,Semicarbazone

(3S)-3-[(1-Methylindole-2-carbonyl)prolinyl] amino-4-oxobutanoic acid,t-butyl ester semicarbazone (50 mg, 0.1 mmol) was dissolved in anisole(0.2 mL) and methylene chloride (2 mL) and the resultant solution wastreated with TFA (1 mL). This reaction mixture was then stirred for 1hour under a nitrogen atmosphere at room temperature. The reactionmixture was concentrated in vacuo and chased with methylene chloride togive a purple film. The film was triturated with ether to afford thetitle product as a purple powder (47 mg). TLC: (methylenechloride:methanol:acetic acid, 20:1:1, silica gel): R_(f)=0.18; ¹H NMR(CD₃ OD): δ 7.63-6.93 (m, 6H), 6.67 (bs, 1H), 4.89-4.50 (m, 2H),3.86-3.74 (m, 5H), 2.82-2.74 (m, 2H), 2.40-2.30 (m, 1H), 2.15-1.90 (m,3H).

Example 7(3S)-3-[(1-Methylindole-2-carbonyl)prolinyl]amino-4-oxobutanoic Acid

(3S)-3-[(1-Methylindole-2-carbonyl)prolinyl] amino-4-oxobutanoic acid,semicarbazone (87 mg, 0.22) mmol) was dissolved in methanol (3 mL),formaldehyde 1 mL, 37% wt. aq) and acetic acid (1 mL) and the resultingmixture was stirred for 4 hours under a nitrogen atmosphere at roomtemperature. The reaction mixture was concentrated in vacuo diluted withwater, and extracted twice with ethyl acetate. The ethyl acetatesolution was washed with brine, dried over sodium sulfate andconcentrated to give brown oil (22 mg) which was triturated with etherto afford the title product as a light brown powder (8 mg). TLC:(methylene chloride:methanol:acetic acid, 20:1:1, silica gel):R_(f)0.28; MS (EI) for C₁₉H₂₁N₃O₅+H⁺=372; C₁₉H₂₁N₃O₅—H⁺=370).

Example 8 (3S)-3-[(1-Methylindole-2-carbonyl)valinyl]amino-4-oxobutanoicAcid, t-Butyl Ester Semicarbazone

1-Methylindole-2-carboxylic acid (88 mg, 0.5 mmol) and(3S)-3-(Valinyl)amino-4-oxobutanoic acid, t-butyl ester semicarbazone(163 mg, 0.5 mmol) were dissolved in DMF (1 mL) and methylene chloride(2 mL) then both DMAP (61 mg, 0.50 mmol) and EDAC (134 mg, 0.7 mmol)were added to the solution under a nitrogen atmosphere at 0° C. Stirringwas continued for 1 hour at 0° C. and an additional 4 hours at roomtemperature. The reaction mixture was partitioned between ethyl acetateand 5% KHSO₄ solution. The ethyl acetate solution was washedsuccessively with 5% KHSO solution, saturated sodium bicarbonatesolution and brine solutions, dried over sodium sulfate, andconcentrated to give a yellow foam. Trituration of the foam with etherafforded the title product as a slightly yellow powder (203 mg, 86%).TLC: (methanol/methylene chloride: 5/95, silica gel): R_(f)=17.

Example 9 (3S)-3-[(1-Methylindole-2-carbonyl)valinyl]amino-4-oxobutanoicAcid Semicarbazone

(3S)-3-[(1-Methylindole-2-carbonyl)valinyl]amino-4-oxobutanoic acid,t-butyl ester semicarbazone (170 mg, 0.36 mmol) was dissolved in anisole(0.2 mL) and methylene chloride (2 mL) and the resulting solution wastreated with TFA (1 mL). The resulting solution was stirred for 3.5hours under a nitrogen atmosphere at room temperature. The reactionmixture was concentrated in vacuo and chased with methylene chloride togive a purple foam. Trituration of the foam with ether afforded thetitle product as a solid purple powder (133 mg, 89%).

Example 10(3S)-3-[(1-Methylindole-2-carbonyl)valinyl]amino-4-oxobutanoic Acid

(3S)-3-[(1-Methylindole-2-carbonyl)valinyl]amino-4-oxobutanoic acid,semicarbazone (136 mg, 0.33 mmol) was dissolved in methanol (3 mL),formaldehyde (1 mL, 37% wt. aq) and acetic acid (1 mL) and the resultingmixture was stirred for 5 hours under a nitrogen atmosphere at roomtemperature. The reaction mixture was concentrated in vacuo diluted withwater, and extracted twice with ethyl acetate. The combined ethylacetate solutions were washed with brine, dried over sodium sulfate andconcentrated in vacuo to give a purple foam which was triturated withether to afford the title product as a purple powder (40 mg, 33%). TLC:(methylene chloride:methanol:acetic acid, 20:1:1, silica gel):R_(f)=0.36; MS (E) for C₁₉H₂₃N₃O₅+H⁺=374; C₁₉H₂₃N₃O₅—H⁺=372)

Example 11(3S)-3-[(1-Methylindole-2-carbonyl)leucinyl]amino-4-oxobutanoic Acid,t-Butyl Ester Semicarbazone

1-Methylindole-2-carboxylic acid (70 mg, 0.4 mmol) and3(S)-(Leucinyl)amino-4-oxobutanoic acid, t-butyl ester semicarbazone(131 mg, 0.4 mmol) were dissolved in methylene chloride (2 mL) and bothDMAP (49 mg, 0.40 mmol) and EDAC (107 mg, 0.56 mmol) were added to thesolution under a nitrogen atmosphere at 0° C. Stirring was continued for1 hour at 0° C. and an additional 3 hours at room temperature. Thereaction mixture was partitioned between ethyl acetate and 5% KHSO₄solution. The ethyl acetate solution was washed successively with 5%KHSO₄ solution, saturated with sodium bicarbonate solution (2×) andbrine, dried over sodium sulfate, and concentrated in vacuo to give acrude solid. Trituration of the solid with ether afforded the titleproduct as a white powder (156 mg, 80%). TLC: (methanol/methylenechloride: 5/95, silica gel): R_(f)=0.42; ¹H NMR (CDCl₃+CD₃OD): δ 8.18(s, 1H), 7.66-7.11 (m, 6H), 6.97 (s, 1H), 6.32 (d, J=7.7, 1H), 4.95-4.88(m, 1H), 4.70-4.62 (m, 1H), 4.03 (s, 3H), 2.82-2.56 (m, 2H), 1.87-1.58(m, 3H), 1.38 (9H), 1.00 (t, J-6.3, 6H).

Example 12(3S)-3-[(1-Methylindole-2-carbonyl)leucinyl]amino-4-oxobutanoic Acid,Semicarbazone

(3S)-3-[(1-Methylindole-2-carbonyl)leucinyl]amino-4-oxobutanoic acid,t-butyl ester semicarbazone (132 mg, 0.27 mmol) was dissolved in anisole(0.2 mL) and methylene chloride (2 mL) and the resulting solution wastreated with TFA (1 mL). The resulting solution was stirred for 3 hoursunder a nitrogen atmosphere at room temperature. The reaction mixturewas concentrated in vacuo and chased with methylene chloride to give apink foam. Trituration of the foam with ether afforded the title productas a pink powder (108 mg, 92%). TLC: (methylene chloride:methanol:aceticacid, 20:1:1, silica gel): R_(f)0.22; ¹H NMR (CD₃ OD): δ 7.62 (dt,J=8.0, 1.1, 1H), 7.45 (dd, J=8.5, 0.8, 1H), 7.32-7.23 (m, 21), 7.13-7.08(m, 2H), 4.94-4.89 (m, 1H), 4.64-4.59 (m, 1H), 3.98 (s, 3H), 2.78 (d,J=6.2, 2H), 1.82-1.70 (m, 3H), 1.02 (d, J=6.0, 3H), 0.99 (d, J=6.3, 3H).

Example 13(3S)-3-[(1-Methylindole-2-carbonyl)leucinyl]amino-4-oxobutanoic Acid

(3S)-3-[(1-Methylindole-2-carbonyl)leucinyl]amino-4-oxobutanoic acid,semicarbazone (90 mg, 0.21 mmol) was dissolved in methanol (3 mL),formaldehyde (1 mL, 37% wt. aq) and acetic acid (1 mL) and the resultingsolution was stirred for 7 hours under a nitrogen atmosphere at roomtemperature. The reaction mixture was concentrated in vacuo, dilutedwith water, and extracted twice with ethyl acetate. The ethyl acetatesolution was washed with brine, dried over sodium sulfate, andconcentrated in vacuo to give a purple foam which was triturated withether to afford the title product as a purple powder (35 mg, 43%). TLC:(methylene chloride:methanol:acetic acid, 20:1:1, silica gel):R_(f)=0.45; MS(EI) for C₂₀H₂₅N₃O₅; M+H⁺=388; M−H⁺=386.

Example 14(3S)-3-[(1-Methylindole-2-carbonyl)phenyalaninyl]amino-4-oxobutanoicAcid, t-Butyl Ester Semicarbazone

1-Methylindole-2-carboxylic acid (72 mg, 0.41 mmol) and3(S)-(phenylalaninyl]amino4-oxobutanoic acid, t-butyl estersemicarbazone (154 mg, 0.41 mmol) were dissolved in methylene chloride(2 mL) and both DMAP (53 mg, 0.43 mmol) and EDAC (109 mg, 0.57 mmol)were added to the solution under a nitrogen atmosphere at 0° C. Stirringwas continued for 1 hour at 0° C. and an additional 4 hours at roomtemperature. The reaction mixture was partitioned between ethyl acetateand 5% KHSO₄ solution, successively, dried over sodium sulfate, andconcentrate to give a white solid. Trituration of the solid with etherafforded the title product as a white powder (179 mg, 82%) TLC:(methanol/methylene chloride: 5/95, silica gel): R_(f)=0.44.

Example 15(3S)-3-[(1-Methylindole-2-carbonyl)phenylalaninyl]amino-4-oxobutanoicAcid, Semicarbazone

(3S)-3-[(1-Methylindole-2-carbonyl)phenylalaninyl]amino-4-oxobutanoicacid, t-butyl ester semicarbazone (154 mg, 0.30 mmol) was dissolved inanisole (0.2 mL) and methylene chloride (2 mL) and the resultingsolution was treated with TFA (1 mL). The resulting solution was stirredfor 4 hours under a nitrogen atmosphere at room temperature. Thereaction mixture was concentrated in vacuo and azeotroped with methylenechloride to give a purple solid. Trituration of the solid with etherafforded the title product as a purple powder (141 mg, 100%). TLC:(methylene chloride:methanol:acetic acid, 20:1:1, silica gel):R_(f)=0.25.

Example 16(3S)-3-[(1-Methylindole-2-carbonyl)phenylalaninyl]amino-4-oxobutanoicAcid

(3S)-3-[(1-Methylindole-2-carbonyl)phenyl-alaninyl]amino-4-oxobutanoicacid, semicarbazone (116 mg, 0.25 mmol) were dissolved in methanol (3mL), formaldehyde (1 mL, 37% wt. aq) and acetic acid (1 mL) and theresulting solution was stirred for 9 hours under a nitrogen atmosphereat room temperature. The reaction mixture was concentrated in vacuodiluted with water, and extracted twice with ethyl acetate. The ethylacetate solution was washed with brine, dried over sodium sulfate andconcentrated to give a crude product which was triturated with ether toafford the title product as a brown powder (26 mg, 25%). TLC: (methylenechloride:methanol:acetic acid, 20:1:1, silica gel): R_(f)=0.33; MS(EI)for C₂₃H₂₁N₃O₅; M+H⁺=422; M−H⁺=420.

Example 17 (1-Methylindole-2-carbonyl)glycine, Methyl Ester

DMAP (1.222 g, 0.01 mol) and EDAC (2.680 g, 0.014 mol) were added assolids to a solution of 1-methylindole-2-carboxylic acid (1.752 g, 0.01mol) and glycine methyl ester hydrochloride (1.256 g, 0.01 mol) inmethylene chloride (30 mL) and DMP (5 mL) under a nitrogen atmosphere at0° C. Stirring was continued for 1 hour at 0° C. and then for 3 hours atroom temperature. The reaction mixture was partitioned with ethylacetate and 5% KHSO₄ solution and the aqueous layer was extracted withethyl acetate. The combined ethyl acetate solution was washed with 5%KHSO4 solution, saturated sodium bicarbonate solution (2×) solution andbrine, dried over sodium sulfate, and concentrated to give a purplepowder as crude product. Trituration of the powder with ether affordedthe title product (1.734 mg, 70%). TLC: (methanol/methylene chloride1:9): R_(f)=0.61; ¹H NMR (CDCl₃): δ 7.65 (dt, J=8.0, 1.1, 1H), 7.41-7.31(m, 2H), 7.16 (dd, J=6.6, 1.4, 1H) 6.96 (d, J=0.5, 1H), 6.67 (bs, 1H),4.25 (d, J=5.2, 2H), 4.05 (s, 3H), 3.82 (s, 3H).

Example 18 (1-Methylindole-2-carbonyl)glycine

(1-Methylindole-2-carbonyl)glycine methyl ester (1.687 g, 6.85 mmol) wasdissolved in 1,4-dioxane (10 mL) and was treated with 1 N lithiumhydroxide (7.0 mL, aq) with stirring. The reaction mixture turned clearimmediately and was acidified with 1N HCl and concentrated to remove1,4-dioxane to result in a purple precipitate. The precipitate wasfiltered, washed with water, and dried in vacuo to give the titleproduct as a purple powder (1.482 g, 93%). TLC: (methylenechloride:methanol:acetic acid, 20:1:1, silica gel): R_(f)=0.28; ¹H NMR(CD₃OD): δ 7.61 (dt, J=8.2, 1H). 7.44 (dd, J=8.5, 0.8, 1H), 7.32-7.26(m, 1H), 7.13-7.09 (m, 1H), 7.04 (s, 1H), 4.08 (s, 2H), 3.99 (s, 3H).

Example 19(3S)-3-[(1-Methylindole-2-carbonyl)glycine]amino-4-oxobutanoic Acid,t-Butyl Ester Semicarbazone

(1-Methylindole-2-carbonyl)glycine (186 mg, 0.8 mmol) was dissolved inmethylene chloride (5 mL) and DMF (1 mL) and the resulting solution wastreated with 1-hydroxybenzotriazole hydrate (129 mg, 0.84 mmol) and EDAC(215 mg, 1.12 mmol) under a nitrogen atmosphere and the reaction mixturestirred for 10 minutes at 0° C. 3(S)-Amino-4-oxobutanoic acid, t-butylester semicarbazone p-toluenesulfate (312 mg, 0.8 mmol) followed byN-methylmorpholine (0.09 mL, 0.8 mmol), were added to the reactionmixture and the mixture was stirred for 1 hour at 0° C. and anadditional 4 hours at room temperature. The reaction mixture waspartitioned between ethyl acetate and 5% KHSO₄, and the productprecipitated out during the work-up. The white precipitate from theaqueous portion was obtained by filtration and washing with water andether. Another portion of white precipitate was obtained byconcentration of the organic layer and trituration of the residue withether. The combined precipitate was the title product (297 mg, 66%).TLC: (methanol/methylene chloride: 1/9, silica gel): R_(f)=0.42; ¹H NMR(CDCl₃) δ 7.65 (d, J=8.0, 1H), 7.41-7.34 (m, 2H), 7.19-7.13 (m, 2H),7.05 (d, J=0.5, 1H), 4.95-4.93 (m, 1H), 4.08 (s, 2H), 4.03 (s, 3H),2.79-2.59 (m, 2H), 1.41 (s, 9H).

Example 20(3S)-3-[(1-Methylindole-2-carbonyl)glycine]amino-4-oxobutanoic Acid,Semicarbazone

(3S)-3-[(1-Methylindole-2-carbonyl)glycinyl]amino-4-oxobutanoic acid,t-butyl ester semicarbazone (118 mg, 0.26 mmol) was dissolved in anisole(0.2 mL) and methylene chloride (2 mL) and the resulting solution wastreated with TFA (1 mL). The resulting solution was stirred for 3 hoursunder a nitrogen atmosphere at room temperature. The reaction mixturewas concentrated in vacuo and chased with methylene chloride to give agreen solid. Trituration of the solid with ether afforded the titleproduct as a green powder (88 mg, 87%). TLC: (methylenechloride:methanol:acetic acid, 20:1:1, silica gel): R_(f)=0.47; ¹H NMR(CD₃ OD): δ 7.63-7.08 (m, 6H), 4.95 (m, 1H), 4.05 (s, 2H), 4.01 (s, 3H),3.77 (d, J=5.8, 2H).

Example 21(3S)-3-[(1-Methylindole-2-carbonyl)glycinyl]-amino-4-oxobutanoic Acid

(3S)-3-[(1-Methylindole-2-carbonyl)glycinyl]amino-4-oxobutanoic acid,semicarbazone (76 mg, 0.20 mmol) was dissolved in a mixture of methanol(3 mL), formaldehyde (1 mL, 37% wt. aq) and acetic acid (1 mL) and themixture was stirred for 6 hours under a nitrogen atmosphere at roomtemperature. The reaction mixture was concentrated in vacuo, dilutedwith water, extracted twice with ethyl acetate. The combined ethylacetate solutions were washed with brine, dried over sodium sulfate, andconcentrated to give a crude product which was triturated with ether toafford the title product as a light yellow powder (29 mg, 44%). TLC:(methylene chloride:methanol:acetic acid, 8:1:1, silica gel):R_(f)=0.61; MS(EI) for C₁₆H₁₇N₃O₅:M+H⁺, 330. ¹H NMR (CD₃OD): δ 7.73-7.08(m, 5H), 4.90-3.8 (m, 7H), 2.72-2.47 (m, 2H).

Example 22(3S)-3-[(1-Benzylindole-2-carbonyl)alaninyl]amino-4-oxobutanoic Acid,t-Butyl Ester Semicarbazone

1-Benzylindole-2-carboxylic acid (477 mg, 1.9 mmol) and3(S)-(alaninyl)amino-4-oxobutanoic acid, t-butyl ester semicarbazone(581 mg, 1.9 mmol) were dissolved in methylene chloride (8 mL) and bothDMAP (232 mg, 1.9 mmol) and EDAC (498 mg, 2.6 mmol) were added to thesolution under a nitrogen atmosphere at 0° C. The resultant solution wasstirred for 1 hour at 0° C. and an additional 2 hours at roomtemperature. The reaction mixture was diluted with ethyl acetate, washedsuccessively with saturated sodium bicarbonate solution and brine, driedover sodium sulfate, and concentrated to give a yellow foam. Flashcolumn chromatographic purification of the foam (silica gel,methanol/methylene chloride 2-5%) afforded the title product as a whitepowder (570 mg, 56%). TLC: (methanol/methylene chloride: 1/9, silicagel): R_(f)=0.38; ¹H NMR (CDCl₃): δ 8.60 (bs, 1H), 7.67 (dd, J=8.0, 1.1,1H), 7.50 (d, J=8.0, 1H), 7.33-7.01 (m, 8H), 6.79 (d, J=7.4, 1H), 5.78(s, 2H), 4.87-4.83 (m, 1H), 4.67-4.62 (m, 1H), 2.73-2.43 (m, 2H), 1.46(d, J=7.1, 3H), 1.39 (s, 9H).

Example 23(3S)-3-[(1-Benzylindole-2-carbonyl)alaninyl]amino-4-oxobutanoic Acid,Semicarbazone

(3S)-3-[(1-Benzylindole-2-carbonyl)alaninyl]amino-4-oxobutanoic acid,t-butyl ester semicarbazone (247 mg, 0.46 mmol) was dissolved in anisole(0.5 mL) and methylene chloride (2 mL) and the resultant mixture wastreated with TFA (1 mL). The resulting solution was stirred for 3.5hours under a nitrogen atmosphere at room temperature. The reactionmixture was concentrated and chased with methylene chloride to give alight green solid. Trituration of the solid with ether afforded thetitle product as a green powder (215 mg, 98%). TLC: (methylenechloride:methanol:acetic acid, 8:1:1, silica gel): R_(f)=0.50; ¹H NMR(CD₃OD): δ 8.26 (d, J=8.0, 1H), 7.65 (d, J=8.0, 1H), 7.39 (dd, J=8.5,0.8, 1H), 7.26-7.01 (m, 8H), 5.69 (d, J=7.4, 2H), 4.56-4.49 (m, 1H),2.77-2.62 (m, 2H), 1.43 (d, J=7.4, 3H).

Example 24(3S)-3-[(1-Benzylindole-2-carbonyl)alaninyl]amino-4-oxobutanoic Acid

(3S)-3-[(1-Benzylindole-2-carbonyl)alaninyl]amino-4-oxobutanoic acid,semicarbazone (176 mg, 0.37 mmol) was dissolved in methanol (4.5 mL),formaldehyde (1.5 mL, 37% wt. aq) and acetic acid (1.5 mL) and theresulting mixture was stirred for 4 hours under a nitrogen atmosphere atroom temperature. The reaction mixture was concentrated in vacuo,diluted with water, and extracted twice with ethyl acetate. The ethylacetate solution was washed with brine, dried over sodium sulfate, andconcentrated to give a crude product which was triturated with ether toafford the title product as a light green powder (113 mg, 72%). TLC:(methylene chloride:methanol:acetic acid, 20:1:1, silica gel):R_(f)=0.38; MS for C₂₃H₂₃N₃O₅; M+H⁺=422; M−H⁺=4.20. ¹H NMR (CD₃OD): δ7.65 (d, J=8.0, 1H), 7.37 (dd, J=8.2, 0.8, 1H), 7.24-7.04 (m, 8H),5.87-5.73 (m, 2H), 4.60-4.49 (m, 2H), 4.32-4.23 (m, 1H), 2.69-2.44 (m,2H), 1.41 (d, J=7.1, 2 sets, 3H).

Example 25(3S)-3-[(1-(4′-Butenyl)indole-2-carbonyl)valinyl]amino-4-oxobutanoicAcid t-Butyl Ester Semicarbazone

[1-(4′-Butenyl)indole]-2-carboxylic acid (108 mg, 0.5 mmol) and3(S)-(valinyl)amino-4-oxobutanoic acid, t-butyl ester semicarbazone (163mg, 0.5 mmol) were dissolved in methylene chloride (3 mL). To thissolution was added both DMAP (61 mg, 0.5 mmol) and EDAC (134 mg, 0.7mmol) under a nitrogen atmosphere at 0° C. and the resultant reactionmixture was stirred for 1 hour at 0° C. and an additional 5 hours atroom temperature. The reaction mixture was diluted with ethyl acetate,washed successively with saturated sodium bicarbonate solution andbrine, dried under sodium sulfate, and concentrated to give a yellowfoam. Trituration of the foam with ether afforded the title product as aslightly yellow powder (146 mg, 55%). TLC: (methanol/methylene chloride:1/9, silica gel): R_(f)=0.23; ¹H NMR (CDCl₃): δ 8.69 (bs, 1H), 7.64 (d,J=8.0, 1H) 7.41-7.13 (m, 3H), 6.99 (s, 1H), 6.91 (d, J=8.8, 1H),5.85-5.71 (m, 1H), 5.04-4.94 (m, 3H), 4.65-4.45 (m, 3H), 3.52-2.50 (m,4H), 2.33-2.26 (m, 1H), 1.41 (s, 9H), 1.05-1.02 (m, 6H).

Example 26(3S)-3-[(1-(4′-Butenyl)indole-2-carbonyl)valinyl]amino-4-oxobutanoicAcid, Semicarbazone

(3S)-3-[(1-(4′-Butenyl)indole-2-carbonyl)valinyl]amino-4-oxobutanoicacid, t-butyl ester semicarbazone (126 mg, 0.24 mmol) was dissolved inanisole (0.2 mL) and methylene chloride (2 mL) and the resultingsolution was treated with TFA (1 mL). The acidified reaction mixture wasstirred for 4 hours under a nitrogen atmosphere at room temperature. Thereaction mixture was concentrated and chased with methylene chloride togive a crude solid. Trituration of the solid with ether afforded thetitle product as a purple powder (99 mg, 88%). TLC: (methylenechloride:methanol:acetic acid, 20:1:1, silica gel): R_(f)=0.36; ¹H NMR(CD₃OD): δ 8.46 (d, J=8.0, 1H) 8.12 (d, J=8.2, 1H), 7.62 (d, J=8.0, 1H),7.46 (dd, J=8.5, 0.8, 1H), 7.31-7.21 (m, 2H), 7.31-7.05 (m, 2H),5.84-5.70 (m, 1H), 4.99-4.78 (m, 3H), 4.62-4.57 (m, 2H), 4.39-4.33 (m,1H), 2.88-2.69 (m, 2H), 2.52 -2.45 (m, 2H), 2.24-2.15 (m, 1H), 1.07-1.02(m, 6H).

Example 27(3S)-3-[(1-(4′-Butenyl)indole-2-carbonyl)valinyl]amino-4-oxobutanoicAcid

(3S)-3-[(−(4′-Butenyl)indole-2-carbonyl)valinyl]amino-4-oxobutanoicacid, semicarbazone (79 mg, 0.17 mmol) was dissolved in methanol (3 mL),formaldehyde (1 mL, 37% wt. aq) and acetic acid (1 mL) and the resultingmixture was stirred for 7 hours under a nitrogen atmosphere at roomtemperature. The reaction mixture was concentrated in vacuo, dilutedwith water, and extracted twice with ethyl acetate. The ethyl combinedacetate solutions were washed with brine, dried over sodium sulfate andconcentrated to give a crude product which was triturated with ether toafford the title product as a light purple powder (24 mg, 34%). TLC:(methylene chloride:methanol:acetic acid, 20:1:1, silica gel):R_(f)=0.60; MS(EI) for C₂₂H₂₇N₃O₅: M+H⁺=414; M−H⁺=412. ¹H NMR (CD₃OD): δ8.09-8.05 (m, 1H), 7.62 (d, J=8.0, 1H), 7.46 (dd, J=8.5, 0.8, 11),7.31-7.25 (m, 1H), 7.13-7.07 (m, 2H), 5.85-5.71 (m, 1H), 4.99-4.90 (m,3H), 4.62-4.54 (m, 3H), 4.41-4.30 (m, 2H), 2.75-2.46 (m, 4H), 2.22-2.14(m, 1H), 1.06-1.02 (m, 6H).

Example 28(3S)-3-[(1-(2′-(1′-t-Butoxy-1′-oxo)ethyl)indole-2-carbonyl)alaninyl]amino-4-oxobutanoicAcid, t-Butyl Ester Semicarbazone

1-[2′-(1′-t-Butoxy-1′-oxo)ethyl]indole-2-carboxylic acid (220 mg, 0.8mmol) and 3(S)-(alaninyl)amino-4-oxobutanoic acid, t-butyl estersemicarbazone (241 mg, 0.8 mmol) were dissolved in methylene chloride (3mL) and DMF (1 mL) and the resulting solution was treated with both DMAP(98 mg, 0.8 mmol) and EDAC (211 mg. 1.1 mmol). The resultant reactionmixture was stirred for 1 hour at 0° C. and then an additional 3 hoursat room temperature to give a white precipitate. The reaction mixturewas concentrated to remove methylene chloride and quenched with 5% KHSO₄solution. The white solid was collected by filtration, washed with waterand ether and dried in vacuo to afford the title product as a whitepowder (297 mg, 66%). TLC: (methanol/methylene chloride: 1/9, silicagel): R_(f)=0.27. ¹H NMR: (CD₃OD) δ 7.65 (d, J=8.0, 1H), 7.41)d, J=8.0,1H), 7.26 (s, 1H) 7.22 (d, J=3.0, 1H), 7.16-7.11 (m, 1H), 5.32 (d,J=2.2, 2H), 4.94-4.89 (m, 1H), 4.54 (q, J=7.1, 1H), 2.76 (d, 2H), 1.48(d, J=7.4, 3H).

Example 29(3S)-3-[(1-(Carboxymethyl)-indole-2-carbonyl)alaninyl]amino-4-oxobutanoicAcid, Semicarbazone

(3S)-3-[(1-(2′-(1′-t-butoxy- 1′-oxo)ethyl)indole-2-carbonyl)]amino-4-oxobutanoic acid, t-butyl ester,semicarbazone (274 mg, 0.51 mmol) in methylene chloride (2 mL) wastreated with TFA (1 mL). The resulting solution was stirred for 2 hoursunder a nitrogen atmosphere at room temperature. The reaction mixturewas concentrated and chased with methylene chloride to give a solid.Trituration of the solid with ether gave the title product as a lightgray powder (262 mg). TLC: (methylene chloride:methanol:acetic acid,8:1:1, silica gel): R_(f)=0.08. ¹H NMR (CD₃OD): δ 7.65 (d, J=8.0, 1H),7.41 (d, J=8.0, 1H), 7.26 (s, 1H), 7.22 (d, J=3.0, 1H), 7.16-7.11 (m,1H), 5.32 (d, J=2.2, 2H), 4.94-4.89 (m, 1H), 4.54 (q, J=7.1, 1H), 2.76(d, 2H), 1.48 (d, J=7.4, 3H).

Example 30(3S)-3-[(1-(Carboxymethyl)indole-2-carbonyl)alaninyl]amino-4-oxobutanoicAcid

(3S)-3-[(1-(Carboxymethyl)indole-2-carbonyl)alaninyl]amino-4-oxobutanoicacid, semicarbazone (241 mg, 0.47 mmol) was dissolved in methanol (3mL), formaldehyde (1 mL, 37% wt. aq) and acetic acid (1 mL) and theresulting solution was stirred for 3 hours under a nitrogen atmosphereat room temperature. The reaction mixture was concentrated in vacuo,diluted with water and extracted twice with ethyl acetate. The combinedethyl acetate solutions were washed with brine, dried under sodiumsulfate and concentrated to give a glassy material which was trituratedwith ether to afford the title product as a slightly yellow powder (114mg, 63%). TLC: (methylene chloride:methanol:acetic acid, 8:1:1, silicagel): R_(f)=0.16. ¹H NMR (CD₃OD): δ 7.65 (d, J=8.0, 1H), 7.40 (d, J=8.2,1H), 7.33-7.27 (m, 1H), 7.24 (s, 1H), 7.16-7.10 (m, 1H), 5.36 and 5.26(AB, J=17.9, 2H), 4.64-4.50 (m, 2H), 4.34-4.20 (m, 1H), 2.72-2.48 (m,2H), 1.45 (d, J=7.14, 3H, 2 sets).

Example 31(3S)-3-[(1-(3′-(1′-t-Butoxy-1′-oxo)propyl)indole-2-carbonyl)alaninyl]amino-4-oxobutanoicAcid, t-Butyl Ester Semicarbazone

1-(3′-(1′-t-Butoxy-1′-oxo)propyl)indole-2-carboxylic acid (147 mg, 0.51mmol) was dissolved in DMF 3 mL) and to the resulting solution was addedboth DMAP (68 mg, 0.56 mmol) and EDAC (140 mg, 0.73 mmol). Stirring wascontinued for 10 minutes under a nitrogen atmosphere at 0° C.(3S)-3-(Alaninyl)amino-4-oxobutanoic acid, t-butyl ester semicarbazone(154 mg, 0.51 mmol) was added to the reaction mixture, and the mixturewas stirred for 1 hour at 0° C. and then an additional 4 hours at roomtemperature. The reaction mixture was partitioned between 5% KHSO₄solution and ethyl acetate. The ethyl acetate solution was washedsuccessively with 5% KHSO₄ solution, saturated sodium bicarbonatesolution (2×) and brine, dried over sodium sulfate, and concentrated togive a foam as crude product. Trituration of the foam with etherafforded the title product as a white powder (161 mg, 55%). TLC:(methanol/methylene chloride: 1/9, silica gel): R_(f)=0.36; ¹H NMR(CD₃OD): 7.62 (d, J=8.0, 1H), 7.50 (d, J=8.2, 1H), 7.29 (t, J-8.2, 1H),7.22 (d, J=3.0, 1H), 7.16 (s, 1H), 7.11 (t, J=7.4, 1H), 4.96-4.90 (m,1H), 4.82-4.72 (m, 2H), 4.56 (q, J=7.1, 1H), 2.78-2.66 (m, 4H), 1.49 (d,J=7.4, 3H), 1.40 (s, 9H, 1.28 (s, 9H).

Example 32(3S)-3-[(1-(2′-Carboxyethyl)indole-2-carbonyl)alaninyl]amino-4-oxobutanoicAcid Semicarbazone

(3S)-3-[(1-(3′-(1′-t-Butoxy-1′-oxo)propyl)indole-2-carbonyl)alaninyl]amino-4-oxobutanoicacid, t-butyl ester semicarbazone (140 mg, 0.24 mmol) was dissolved inanisole (0.2 mL) and methylene chloride (2 mL) and the suspension wastreated with TFA (1 mL). The resulting solution was stirred for 2 hoursunder a nitrogen atmosphere at room temperature. The reaction mixturewas concentrated and chased with methylene chloride to give a solid.Trituration of the solid with ether gave the title product as acolorless powder (107 mg, 95%). TLC: (methylene chloride:methanol:aceticacid, 8:1:1, silica gel): R_(f)=0.17; ¹H NMR (CD₃OD): δ 7.62 (d, J=8.0,1H), 7.50 (d, J=8.2, 1H), 7.32-7.27 (m, 1H), 7.23 (d, J=3.0, 1H),7.13-7.08 (m, 2h), 4.97-4.90 (m, 1H), 4.80-4.69 (m, 1H), 4.54 (q, J=7.1,1H), 2.82-2.73 (m, 4H), 1.49 (d, J=7.1, 3H).

Example 33(3S)-3-[(1-(2′-Carboxyethyl)indole-2-carbonyl)alaninyl]amino-4-oxobutanoicAcid

(3S)-3-[(1-(2′-Carboxyethyl)indole-2-carbonyl)alaninyl]amino-4-oxobutanoicacid, semicarbazone (95 mg, 0.21 mmol) was dissolved in methanol (3 mL),formaldehyde (1 mL, 37% wt. aq) and acetic acid (1 mL) and the resultantsolution was stirred for 4 hours under a nitrogen atmosphere at roomtemperature. The reaction mixture was concentrated to remove methanol,diluted with water and extracted twice with ethyl acetate. The combinedethyl acetate solutions were washed with brine, dried over sodiumsulfate and concentrated to give a glassy material which was trituratedwith ether to afford the title product as a slightly yellow powder (20mg, 20%). TLC: (methylene chloride:methanol:acetic acid, 8:1:1, silicagel): R_(f)=0.26; ¹H NMR (CD₃OD): δ 7.62 (d, J=8.0, 1H), 7.51 (d, J=1H),7.32-7.27 (m, 1H), 7.13-7.08 (m, 2H), 4.80-4.76 (m, 2H), 4.68-4.52 (m,2H), 4.37-4.25 (m, 1H), 2.84-2.50 (m, 3H1), 1.47 (d, J=7.1, 3H, 2 sets).

Example 34 2,6-Dichlorobenzyloxyethanol

Sodium hydride (1.76 g, 0.044 mol, 60% wt. in mineral oil) was slowlyadded to a solution of ethylene glycol (11.2 mL) in dry THF (100 mL).The resultant mixture was stirred briefly under a nitrogen atmosphere atroom temperature. α-Bromo-2,6-dichlorotoluene (9.894 g, 0.04 mol) wasadded to the mixture and the mixture was stirred for an additional 5.5hours under a nitrogen atmosphere at room temperature. Additional sodiumhydride (0.400 g) was added and the mixture was then stirred for 24hours at room temperature. The reaction mixture was concentrated toremove THF, and the residue was partitioned between ether and water. Theaqueous layer was back extracted with ether (2×). The combined organicsolution was washed with water and brine, dried over sodium sulfate,filtered and concentrated to give a crude oil. The oil was flashchromatographed on silica gel with ethyl acetate/hexanes (10-50%) togive the title product as a yellow oil (4.56 g, 51%). TLC: (ethylacetate/hexanes, 30/70): R_(f)=0.26. ¹H NMR (CDCl₃): δ 7.35-7.18 (m,3H), 4.84 (s, 2H), 3.76-3.66 (m, 4H).

Example 35

5-(2′,6′-Dichlorobenzyloxy)-4-hydroxy-3-nitropentanoic Acid, T-ButylEster

DMSO was added dropwise to a solution of (47.5 mL) oxalyl chloride (7.5mL, 15.0 mmol, 2.0 M in methylene chloride) and the resultant reactionmixture was stirred for 10 min at −78° C. 2,6-Dichlorobenzyloxyethanol(2211 mg, 10 mmol) in dry methylene chloride (5 mL) was added dropwiseto the mixture and the mixture was then stirred for 15 minutes under anitrogen atmosphere at −78° C. Triethylamine (8.4 mL, 60 mmol) was addeddropwise to the reaction mixture, and the resultant mixture was stirredfor 10 min at −78° C., then allowed to warm to 0° C. (over a period ofapproximately 20 min). A methylene chloride solution of tert-butyl3-nitropropionate (1927 mg, 11.0 mmol in 5 mL of dry methylene chloride)was added dropwise to the reaction mixture and the mixture was stirredfor 1 hour. The residue was extracted with ether and the resultant whitesolid was collected by filtration. The organic filtrate was washed with5% KHSO₄ solution (2×) and brine, dried over sodium sulfate, andconcentrated to give a crude oil (3.95 g). The oil was subjected toflash chromatography on silica gel with ethyl acetate/hexanes (1:2) toafford the title product as a yellow oil (2.917 g, 74%). TLC: (ethylacetate, hexanes, 60/40): R_(f)=0.54.

Example 36 3-Amino-5-(2′,6′-dichlorobenzyloxy)-4-hydroxypentanoic Acid,T-Butyl Ester

A mixture of 5-(2′,6′-dichlorobenzyloxy)-4-hydroxy-3-nitropentanoic acidt-butyl ester (2.213 g, 0.0056 mol) and wet Raney nickel (3.4 g) inmethanol (150 mL) was stirred for 2 hours under a hydrogen balloon atroom temperature. The reaction mixture was filtered through Celite andthe filter cake was washed with methanol. The filtrate was concentratedand chased with methylene chloride to give the title product (2.078 g,100%). TLC: (methanol/methylene chloride 1/9): R_(f)=0.21.

Example 37

N-(1,3-Dimethylindole-2-Carbonyl)Valine DMAP (367 mg, 3.0 mmol) and EDAC(748 mg, 3.9 mmol) were added as solids to a solution of1,3-dimethylindole-2-carboxylic acid (568 mg, 3.3 mmol) in DMF (5 mL),and the resultant mixture was stirred for 10 minutes under a nitrogenatmosphere at 0° C. A methylene chloride solution of the methyl ester ofvaline (553 mg, 3.3 mmol, in 5 mL of methylene chloride) was added tothe mixture, and the mixture was first stirred for one hour at 0° C.then for 5 hours at room temperature. The reaction mixture waspartitioned between ethyl acetate and 5% KHSO₄ solution and the aqueoussolution was back-extracted with ethyl acetate. The combined ethylacetate washes were in turn washed with 5% KHSO₄ solution saturatedsodium bicarbonate solution (2×) and brine, dried over sodium sulfate,and concentrated to give the title product as a yellow syrup (900 mg).

A 1,4-dioxane solution (5 mL) of the above yellow syrup was treated withan aqueous solution of lithium hydroxide (1.0 M LiOH, 3.0 mL) and theresultant mixture was stirred for 1 hour at room temperature (themixture became homogeneous). The reaction mixture was acidified with 1 Mhydrochloric acid and extracted with ethyl acetate (3×). The combinedethyl acetate solutions were washed with brine, dried over sodiumsulfate, and concentrated to give the title product as a yellow foam(839 mg). ¹H NMR (CD₃OD): δ 7.58 (dt, J=8.0, 0.8, 1H), 7.37 (dd, J-8.0,0.8, 1H), 7.29-7.24 (m, 1H), 7.12-7.06 (m, 1H), 4.57 (d, J=5.8, 1H),3.80 (s, 3H), 2.48 (s, 3H), 3.34-2.28 (m, 1H), 1.10 (d, J=6.9, 3H), 1.07(d, J=6.9, 3H).

Example 38N-[(1,3-Dimethylindole-2-carbonyl)valinyl]-3-amino-4-hydroxy-5-(2′,6′-dichlorobenzyloxy)pentanoicAcid, t-Butyl Ester

1-Hydroxybenzotriazole hydrate (153 mg, 1.0 mmol) and EDAC (268 mg, 1.4mmol) were added to a methylene chloride solution ofN-(1,3-dimethylindole-2-carbonyl)valine (288 mg, 1.0 mmol, in 3 mL ofmethylene chloride). The resultant mixture was stirred for 10 minutesunder a nitrogen atmosphere at room temperature. A methylene chloridesolution of 3-amino-5-(2′,6′-dichlorobenzyloxy)-4-hydroxypentanoic acid,t-butyl ester (364 mg, 1.0 mmol, in 2 mL of methylene chloride) wasadded to the reaction mixture and the mixture was first stirred for onehour under a nitrogen atmosphere at 0° C., and then for 16 hours at roomtemperature. The reaction mixture was partitioned between ethyl acetateand 5% KHSO₄ solution and the aqueous solution was back-extracted withethyl acetate. The combined ethyl acetate solutions were washed with 5%KHSO₄ solution, saturated sodium bicarbonate solution (2×) and brine,dried over sodium sulfate, and concentrated to give crude product (583mg). The crude product was subjected to flash chromatography on silicagel with ethyl acetate/hexane (2/3) to give the title product as a whitesolid (260 mg). TLC: (ethyl acetate/hexanes 1:1): R_(f)=0.38.

Example 39N-[(1,3-Dimethylindole-2-carbonyl)valinyl]-3-amino-4-oxo-5-(2′,6′-dichlorobenzyloxy)pentanoicAcids t-Butyl Ester

Dess-Martin periodinane (195 mg) was added as a solid to a solution ofN-[(1,3-dimethylindole-2-carbonyl)valinyl]-3-amino-4-hydroxy-5-(2′,6′-dichlorobenzyloxy)pentanoicacid, t-butyl ester (96 mg) in DMSO (1.5 ml). The resulting solution wasstirred under a nitrogen atmosphere at room temperature for thirtyminutes, then partitioned between EtOAc and water. The organic phase waswashed with water (2×) and brine, dried (Na₂SO₄), and concentrated togive a white solid (83 mg). Flash chromotographic purification withEtOAc/hexanes (1:1) afforded the title product as a white solid (54 mg).TLC (EtOAc/hexanes; 1:1, silica gel): R_(f)=0.52.

Example 40N-[(1,3-Dimethylindole-2-carbonyl)valinyl]-3-amino-4-oxo-5-(2′,6′-dichlorobenzyloxy)pentanoicAcid

A solution ofN-[(1,3-Dimethylindole-2-carbonyl)valinyl]-3-amino-4-oxo-5-(2′,6′-dichloro-benzyloxy)pentanoicacid, t-butyl ester (49 mg) in anisole (0.2 mL) and methylene chloride(2 mL) was treated with TFA (1 mL) and stirred for 30 minutes under anitrogen atmosphere at room temperature. The resultant solution wasconcentrated and chased with methylene chloride to give a white solid asthe crude product. The crude product was triturated with ether to yieldthe title product as a white powder (34 mg). MS(EI) for C₂₈H₃₁CL₂N₃O₆;MH⁺=576/578; (MH)⁻=574/576.

Example 41N-[(1,3-Dimethylindole-2-carbonyl)valinyl]-3-amino-4-hydroxy-5-fluoropentanoicAcid, t-Butyl Ester

4-Dimethylaminopyridine (DMAP) (67 mg, 0.55 mmol) and1-(3′-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDAC) (125mg, 0.65 mmol) were added as solids to a DMF solution of1,3-dimethylindole-2-carboxylic acid (95 mg, 0.5 mmol in 1 mL of DMF),and the resultant reaction mixture was stirred for 10 minutes under anitrogen atmosphere at 0° C. A methylene chloride solution ofN-(valinyl)-3-amino-4-hydroxy-5-fluoropentanoic acid, t-butyl ester (153mg, 0.5 mmol in 1 mL of methylene chloride) was added and the resultantreaction mixture was first stirred for 1 hour at 0° C. and then for 4hours at room temperature. The reaction mixture was partitioned betweenethyl acetate and 5% KHSO₄ solution and the aqueous solution wasback-extracted with ethyl acetate. The combined ethyl acetate solutionswere washed with 5% KHSO₄ solution, saturated sodium bicarbonatesolution (2×), and brine, dried over sodium sulfate, and concentrated togive a solid. The solid was triturated with ether/hexane to yield thetitle product as a white solid (134 mg, 56%). TLC: (ethylacetate/hexanes, 2:1): R_(f)=0.42. ¹H NMR (CDCl₃): δ 7.59 (d, J=8.8,1H), 7.37 (d, J=7.7, 1H), 7.29-7.24 (m, 1 H), 7.12-7.07 (m, 1H),4.49-4.26 (m, 5H), 3.81-3.79 (m, 3H), 2.66-2.47 (m, 5H), 2.22-2.10 (m, 1H), 1.45-1.41 (m, 9 H), 1.09-1.03 (m, 6 H).

Example 42N-[(1,3-Dimethylindole-2-carbonyl)valinyl]-3-amino-4-oxo-5-fluoropentanoicAcid t-Butyl Ester

Dimethyl sulfoxide (0.09 mL, 1.25 mmol) was added to a solution ofoxalyl chloride (0.19 mL, 2.0 M, 0.38 mmol) in methylene chloride (4mL), and the resultant mixture was stirred for 10 minutes under anitrogen atmosphere at −78° C. A dry methylene chloride solution ofN-[1,3-dimethylindole-2-carbonyl)valinyl]-3-amino-4-hydroxy-5-fluoropentanoicacid, t-butyl ester (119 mg, 0.25 mmol in 1 mL of dry methylenechloride), was added dropwise to the mixture and the resultant reactionmixture was stirred for 15 min at −78° C. Triethylamine (0.21 mL, 1.5mmol) was added dropwise, and the reaction mixture was then stirred for10 minutes at −78° C. then was allowed to warm to room temperature. Thereaction mixture was partitioned between ethyl acetate and 5% KHSO₄solution and the aqueous layer was back-extracted with ethyl acetate.The combined ethyl acetate solutions were washed with 5% KHSO₄ solutionand brine, dried over sodium sulfate, and concentrated to give a crudeproduct. The crude product was chromatographed with ethylacetate/hexanes (2:1) on silica gel gave the title product as a whitesolid (48 mg, 41%). TLC: (ethyl acetate/hexanes, 2:1): R_(f)=0.58.

Example 43N-[(1,3-Dimethylindole-2-carbonyl)valinyl]-3-amino-4-oxo-5-fluoropentanoicAcid

A solution ofN-[(1,3-dimethylindole-2-carbonyl)valinyl]-3-amino-4-oxo-5-fluoropentanoicacid, t-butyl ester (40 mg) in anisole (0.2 mL) and methylene chloride(2 mL) was treated with trifluoroacetic acid (1 mL), and the resultantreaction mixture was stirred for 30 minutes under a nitrogen atmosphereat room temperature. The reaction mixture was concentrated and chasedwith methylene chloride to give a solid. The solid was triturated withether to yield the title product as a brown powder (17 mg). TLC:(methylene chloride/methanol/acetic acid, 20:1:1): R_(f)=0.40. MS (El)for C₁₂H₂₆FN₃O₅: MH⁺=420; MH⁻=418.

Example 44N-[(1-Methylindole-2-carbonyl)valinyl]-3-amino-4-hydroxy-5-fluoropentanoicAcid, t-Butyl Ester

DMAP (95 mg, 0.78 mmol) and EDAC (200 mg, 1.04 mmol) were added as solidto a solution of 1-methylindole-2-carboxylic acid (130 mg, 0.74 mmol)and N-(valinyl)-3-amino-4-hydroxy-5-fluoropentanoic acid, t-butyl ester(227 mg, 0.74 mmol) in methylene chloride (5 mL), and the resultantsolution was stirred for 1 hour under a nitrogen atmosphere at 0° C. andthen 4 hours at room temperature. The reaction mixture was partitionedbetween ethyl acetate and 5% KHSO₄ solution and the aqueous solution wasback-extracted with ethyl acetate. The combined ethyl acetate solutionswere washed with 5% KHSO₄ solution, saturated sodium bicarbonatesolution (2×) and brine, dried over sodium sulfate, and concentrated togive a foam. The foam was triturated with ether to yield the titleproduct as a slightly brown solid (224 mg, 65%). TLC:(methanol/methylene chloride, 1:9): R_(f)=0.46.

Example 45N-[(3-Chloro-1-methylindole-2-carbonyl)valinyl]-3-amino-4-oxo-5-fluoropentanoicAcids t-Butyl Ester

DMSO (0.06 mL, 0.9 mmol) was added to a solution of oxalyl chloride(0.14 mL, 2.0 M, 0.28 mmol, in 4 mL of methylene chloride) and thesolution was then stirred for 10 minutes under a nitrogen atmosphere at−78° C. A solution of N-[(1-methylindole-2-carbonyl)valinyl]-3-amino-4-hydroxy-5-fluoropentanoic acid, t-butyl ester (85 mg,0.18 mmol) in dry methylene chloride (1 mL), was added dropwise to thereaction mixture and the mixture was stirred for 15 minutes at −78° C.Triethylamine (0.15 mL, 1.08 mmol) was added dropwise to the reactionmixture and the mixture was stirred for 10 minutes at −78° C. and thenwas allowed to warm to room temperature. The reaction mixture waspartitioned between ethyl acetate and 5% KHSO₄ solution and the aqueouslayer was back-extracted with ethyl acetate. The combined ethyl acetatesolutions were washed with 5% KHSO₄ solution and brine, dried oversodium sulfate, and concentrated to give a brown foam. The foam wastriturated with ether to afford the title product as a light brownpowder (64 mg). MS for C₂₄H₃₁ClFN₃O₅: (MH)⁻=494/496.

Example 46N-[(3-Chloro-1-Methylindole-2-carbonyl)valinyl]-3-amino-4-oxo-5-fluoropentanoicAcid

A solutionN-[(3-chloro-1-methylindole-2-carbonyl)valinyl]-3-amino-4-oxo-5-fluoropentanoicacid, t-butyl ester (47 mg) in anisole (0.2 mL) and methylene chloride(2 mL) was treated with TFA (1 mL) and the resultant reaction mixturewas stirred for 1 hour under a nitrogen atmosphere at room temperature.The reaction mixture was concentrated and chased with methylenechloride, then triturated with ether to afford a brown powder (28 mg).The powder was subjected to flash chromatography on silica gel withmethanol/methylene chloride containing a drop of acetic acid to give thetitle product (25 mg). TLC: (methylene chloride/methanol, 9:1):R_(f)=0.29. MS(EI) for C₂₀H₂₃ClFN₃O₅: MH⁺=440.442; (M−H)⁻=438/440.

Example 47 N-[(5-Fluoro-1-Methylindole-2-carbonyl)valinyl]-3-amino-4-hydroxy-5-fluoropentanoic Acids t-Butyl Ester

DMAP (257 mg, 2.08 mmol) and EDAC (427 mg, 2.23 mmol) were added assolids to a solution of 5-fluoro-1-methylindole-2-carboxylic acid (359mg, 86 mmol in 3 mL of DMF), and the resultant reaction mixture wasstirred for 10 minutes under a nitrogen atmosphere at 0° C.N-(Valinyl)-3-amino-4-hydroxy-5-fluoropentanoic acid, t-butyl ester (579mg, 1.86 mmol) in DMF (3 mL) was added and the resulting solution wasstirred for 1 hour under a nitrogen atmosphere at 0° C. and 4 hours atroom temperature. The reaction mixture was partitioned between ethylacetate and 5% KHSO₄ solution and the aqueous solution wasback-extracted with ethyl acetate. The combined ethyl acetate solutionswere washed with 5% KHSO₄ solution, saturated sodium bicarbonatesolution (2×) and brine, dried over sodium sulfate, and concentrated togive the title product as a slightly yellow solid (0.827 mg). TLC:(methanol/methylene chloride, 1:9): R_(f)=0.52.

Example 48N-[(3-Chloro-5-fluoro-1-Methylindole-2-carbonyl)valinyl]-3-amido-4-oxo-5-fluoropentanoicAcid, t-Butyl Ester

DMSO (0.60 mL, 8.5 mmol) was added to a methylene chloride solution ofoxalyl chloride (2.1 mL, 2.0 M, 4.2 mmol, in 15 mL of methylenechloride), and the resultant reaction mixture was stirred for 10 minutesunder a nitrogen atmosphere at −78° C. A methylene chloride solution ofN-[(5-fluoro-1-methylindole-2-carbonyl)valinyl]-3-amino-4-hydroxy-5-fluoropentanoicacid, t-butyl ester (820 mg, 1.7 mmol, in 8 mL of dry methylenechloride), and DMSO (0.4 mL) were added dropwise to the reaction mixtureand stirred for 15 minutes at −78° C. TEA (1.4 mL, 10.2 mmol) was addedto the mixture dropwise and the mixture was stirred for 10 minutes at−78° C., then was allowed to warm to room temperature. The reactionmixture was partitioned between ethyl acetate and 5% KHSO₄ solution andthe aqueous layer was back-extracted with ethyl acetate. The combinedethyl acetate solutions were washed with 5% KHSO₄ solution and brine,dried over sodium sulfate, and concentrated to give the title product asa slightly yellow solid. Trituration with ether afforded the titleproduct as a white powder (705 mg, 85%). TLC: (methanol/methylenechloride, 1:9): R_(f)=0.63. MS for C₂₄H₃₀CIF₂N₃O₅: MH⁺=514/516;(M−H)⁻=514.

Example 49N-[(3-Chloro-5-fluoro-1-Methylindole-2-carbonyl)valinyl]-3-amino-4-oxo-5-fluoropentanoicAcid

A solution ofN-[(3-chloro-5-fluoro-1-methylindole-2-carbonyl)valinyl]-3-amino-4-oxo-5-fluoropentanoicacid, t-butyl ester (682 mg) in anisole (1 mL) and methylene chloride(10 mL) was treated with TFA (5 mL), and the resultant reaction mixturewas stirred for 45 minutes under a nitrogen atmosphere at roomtemperature. The reaction mixture was concentrated and chased withmethylene chloride, then triturated with ether to afford the titleproduct as a white powder (500 mg). MS (EI) for C₂₀H₂₂ClF₂N₃O₅:MH⁺=458/460; (M−H)⁻=456/458.

Example 50N-[(1-(3′-phenylpropyl)indole-2-carbonyl)valinyl]-3-amino-4-hydroxy-5-fluoropentanoicAcid, t-Butyl Ester

DMAP (122 mg, 1.0 mmol) and EDAC (249 mg 1.3 mmol) were added as solidsto a DMF solution of 1-(3′-phenylpropyl)indole-2-carboxylic acid (279mg, 1.0 mmol, 2 mL in DMF), and the resultant mixture was stirred for 10minutes under a nitrogen atmosphere at 0° C. A methylene chloridesolution of N-(valinyl)-3-amino-4-hydroxy-5-fluoropentanoic acid,t-butyl ester (306 mg, 1.0 mmol in 2 mL of methylene chloride) was addedto the reaction mixture and the mixture was stirred for 1 hour under anitrogen atmosphere at 0° C. and then 4 hours at room temperature. Theyellow reaction mixture was partitioned between ethyl acetate and 5%KHSO₄ solution and the aqueous solution was back-extracted with ethylacetate. The combined ethyl acetate solutions were washed with 5% KHSO₄solution, saturated sodium bicarbonate solution (2×) and brine, driedover sodium sulfate, and concentrated to give a crude solid (0.827 g).The crude solid was subjected to flash chromatography on silica geleluting with ethyl acetate/hexanes (1:2) afforded the title product as aslightly yellow solid (171 mg). TLC: (ethyl acetate/hexanes 2:1):R_(f)=0.57.

Example 51N-[(1-(3′-Phenylpropyl)indole-2-carbonyl)valinyl]-3-amino-4-oxo-5-fluoropentanoicAcid, t-Butyl Ester

DMSO (0.11 mL, 1.5 mmol) was added to a methylene chloride solution ofoxalyl chloride (0.22 mL, 2.0 M, 0.44 mmol in 3.5 mL in methylenechloride), and the resultant solution was stirred for 10 minutes under anitrogen atmosphere at −78° C. A methylene chloride solution ofN-[(1-(3′-phenylpropyl)indole-2-carbonyl)valinyl]-3-amino-4-hydroxy-5-fluoropentanoicacid, t-butyl ester (169 mg, 0.3 mmol in 1.5 mL of dry methylenechloride) was added dropwise and the resulting solution stirred for 15minutes at −78° C. Triethylamine (0.25 mL, 1.8 mmol) was added dropwiseto the reaction mixture and the mixture was stirred for 10 minutes at−78° C., then was allowed to warm to room temperature. The reactionmixture was partitioned between ethyl acetate and 5% KHSO₄ solution andthe aqueous layer was back-extracted with ethyl acetate. The combinedethyl acetate solutions were washed with 5% KHSO₄ solution and brine,dried over sodium sulfate, and concentrated to give a crude product. Thecrude product was triturated with hexanes to yield the title product asa slightly yellow powder (129 mg, 77%). TLC: (ethyl acetate/hexanes2:1): R_(f)=0.69.

Example 52N-[(1-(3′-phenylpropyl)indole-2-carbonyl)valinyl]-3-amino-4-oxo-5-fluoropentanoicAcid

A solution ofN-[(1-(3′-phenylpropyl)indole-2-carbonyl)valinyl]-3-amino-4-oxo-5-fluoropentanoicacid, t-butyl ester (97 mg) in anisole (0.2 mL) and methylene chloride(2 mL) was treated with TFA (1 mL), and the resultant reaction mixturewas stirred for 1 hour under a nitrogen atmosphere at room temperature.The reaction mixture was concentrated and chased with methylenechloride, then triturated with ether to yield the title product as aslightly yellow powder (44 mg). TLC: (methylene chloride/methanol/aceticacid, 20:1:1): R_(f)=0.4; MS (EI) for C₃₂H₄₀FN₃O₅: MH⁺=510; (M−H)⁻=508.

Example 53N-[(1-Phenylindole-2-carbonyl)valinyl]-3-amino-4-hydroxy-5-fluoropentanoicAcid, t-Butyl Ester

DMAP (122 mg, 1.0 mmol) and EDAC (249 mg, 1.3 mmol) were added as solidsto a DMF solution of 1-phenylindole-2-carboxylic acid (237 mg, 1.0 mmolin 2 mL DMF), and the resultant reaction mixture was stirred for 10minutes under a nitrogen atmosphere at 0° C. A methylene chloridesolution of N-(valinyl)-3-amino-4-hydroxy-5-fluoropentanoic acid,t-butyl ester (306 mg, 1.0 mmol in 2 mL of methylene chloride) was addedto the reaction mixture and the mixture was stirred for 1 hour under anitrogen atmosphere at 0° C. and 4 hours at room temperature. The yellowreaction mixture was partitioned between ethyl acetate and 5% KHSO₄solution and the aqueous solution was back-extracted with ethyl acetate.The combined ethyl acetate solutions were washed with 5% KHSO₄ solution,saturated sodium bicarbonate solution (2×) and brine, dried over sodiumsulfate, and concentrated to give a colorless film (0.827 g). The filmwas subjected to flash chromatography on silica gel with ethylacetate/hexanes (1:2) to yield the title product as a white foam (400mg, 78%). TLC: (ethyl acetate/hexanes 1:1): R_(f)=0.27.

Example 54N-[(1-Phenylindole-2-carbonyl)valinyl]-3-amino-4-oxo-5-fluoropentanoicAcid, t-Butyl Ester

DMSO (0.13 mL, 1.9 mmol) was added to a methylene chloride solution ofoxalyl chloride (0.29 mL, 2.0 M, 0.58 mmol in 4 mL of methylenechloride), and the resultant solution was stirred for 10 minutes under anitrogen atmosphere at 78° C. A methylene chloride solution ofN-[(1-phenylindole-2-carbonyl)valinyl]-3-amino-4-hydroxy-5-fluoropentanoicacid, t-butyl ester (200 mg, 0.38 mmol in 2 mL of dry methylenechloride) was added dropwise and resulting mixture stirred for 15minutes at −78° C. Triethylamine (0.30 mL, 2.1 mmol) was added dropwiseto the mixture, and the resultant mixture was stirred for 10 minutes at−78° C., then was allowed to warm to room temperature. The reactionmixture was partitioned between ethyl acetate and 5% KHSO₄ solution andthe aqueous layer was back-extracted with ethyl acetate. The combinedethyl acetate solutions were washed with 5% KHSO₄ solution and brine,dried over sodium sulfate, and concentrated to give a crude product. Thecrude product was triturated to yield the title product as a slightlyyellow powder (181 mg). TLC: (ethyl acetate/hexanes 1:1): R_(f)=0.43.

Example 55N-[(1-Phenylindole-2-carbonyl)valinyl]-3-amino-4-oxo-5-fluoropentanoicAcid

A solution of N-[(1-phenylindole-2-carbonyl)valinyl]-3-amino-4-oxo-5-fluoropentanoic acid, t-butyl ester (154 mg) inanisole (0.2 mL) and methylene chloride (2 mL) was treated with TFA (1mL), and the resultant reaction mixture was stirred for one hour under anitrogen atmosphere at room temperature. The reaction mixture wasconcentrated and chased with methylene chloride, then triturated withether to yield the title product as a white powder (100 mg). TLC:(methylene chloride/methanol/acetic acid, 20:1:1): R_(f)=0.38, MS (EI)for C₂₅H₂₆FN₅O₅: MH⁺=468; (M−H)⁻=466.

Example 56N-[1-(2′-((1′-t-Butoxy-1′-oxo)ethyl)indole-2-carbonyl)valinyl]-3-amino-4-hydroxy-5-fluoropentanoicAcid, t-Butyl Ester

DMAP (122 mg, 1.0 mmol) and EDAC (249 mg, 1.3 mmol) were added as solidsto a DMF solution of(1-(2′-((1′-t-butoxy-1′-oxo)ethyl)indole-2-carboxylic acid (275 mg, 1.0mmol in 2 mL of DMF), and the resultant solution was stirred for 10minutes under a nitrogen atmosphere at 0° C. A methylene chloridesolution of N-(valinyl)-3-amino-4-hydroxy-5-fluoropentanoic acid,t-butyl ester (306 mg, 1.0 mmol in 2 mL of methylene chloride) was addedto it, stirred for 1 hour under a nitrogen atmosphere at 0° C. and 4hours at room temperature. The yellow reaction mixture was partitionedbetween ethyl acetate and 5% KHSO₄ solution and the aqueous solution wasback-extracted with ethyl acetate. The combined ethyl acetate solutionswere washed with 5% KHSO₄ solution, saturated with sodium bicarbonatesolution (2×) and brine, dried over sodium sulfate, and concentrated togive a colorless film (0.827 g). The film was flash chromatographed onsilica gel with ethyl acetate/hexane (1:1) to yield the title product asa white foam (461 mg). TLC: (ethyl acetate/hexanes 30:70): R_(f)=0.11.

Example 57N-[(1-(2′-((1′-t-Butoxy-1′-oxo)ethyl)indole-2-carbonyl)valinyl]-3-amino-4-Oxo-5-fluoropentanoicAcid, t-Butyl Ester

A mixture ofN-[(1-(2′-((1′-t-butoxy-1′-oxo)ethyl)indole-2-carbonyl)valinyl]-3-amino-4-hydroxy-5-fluoropentanicacid, t-butyl ester (230 mg, 0.41 mmol), N-methylmorpholine N-oxide (71mg, 0.61 mmol) and powdered molecular sieves (205 mg) in dry methylenechloride (2 mL) was stirred for 1.5 hours under a nitrogen atmosphere atroom temperature. Tetra(propyl)ammonium perruthenate (7 mg) was addedand the resulting mixture was stirred for 2 hours under a nitrogenatmosphere at room temperature. The reaction mixture was filteredthrough silica gel with ethyl acetate as the eluent. The filtrate wasconcentrated and chromatographed on silica gel with ethylacetate/hexanes (approximately 1:2 to approximately 1:1) to yield thetitle product as a yellow oil (100 mg). TLC: (ethyl acetate/hexanes30/70): R_(f)=0.27.

Example 58

N-[(1-(Carboxymethyl)indole-2-carbonyl)valinyl]-3-amino-4-oxo-5-fluoropentanoic Acid

A solution ofN[(1-(2′-((1′-t-butoxy-1′-oxo)ethyl)indole-2-carbonyl)valinyl]-3-amino-4-oxo-5-fluoropentanoicacid, t-butyl ester (100 mg) in anisole (0.2 mL) and methylene chloride(2 mL) was treated with TFA (1 mL). The resultant reaction mixture wasstirred for 30 minutes under a nitrogen atmosphere at room temperature.The reaction mixture was concentrated and chased with methylenechloride, then triturated with ether to yield the title product as alight yellow powder (26 mg). TLC: (methylene chloride/methanol, 8:1:1):R_(f)=0.32. MS (El) for C₂₁H₂₄FN₃O₇: MH⁺=450; (M−H)⁻=448.

Example 59N-[(1-Methylindole-2-carbonyl)valinyl]-3-amino-4-hydroxy-5-fluoropentanoicAcid, t-Butyl Ester

To a solution of 1-methylindole-2-carboxylic acid (130 mg, 0.74 mmol)and N-(valinyl)-3-amino-4-hydroxy-5-fluoropentanoic acid, tert-butylester in methylene chloride (5 mL) and cooled to 0° C. Solid4-dimethylaminopyridine (DMAP) (95 mg, 0.78 mmol) and1-(3′-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDAC) (200mg, 1.04 mmol) were added to the solution at 0° C. The reaction mixturewas stirred at 0° C. for 1 h and allowed to warm slowly to roomtemperature. After 4 h the reaction was partitioned between ethylacetate (EtOAc) and 5% KHSO₄ aqueous solution. The organic layer waswashed with 5% KHSO₄ solution, saturated sodium bicarbonate solution,brine, dried (Na₂SO₄) and concentrated to a foam. The crude residue wastriturated with diethyl ether and the solid filtered to afford the titlecompound as a light brown solid (224 mg, 65% yield). TLC(MeOH:CH₂Cl₂,1:9): R_(f)=0.46.

Example 60N-[(1-Methylindole-2-carbonyl)valinyl]-3-amino-4-oxo-5-fluoropentanoicAcid, t-Butyl Ester

To a solution ofN-[(1-methylindole-2-carbonyl)valinyl]-3-amino-4-hydroxy-5-fluoropentanoicacid, t-butyl ester (51 mg, 0.11 mmol) in DMSO(1 mL) was addedDess-Martin periodinane (110 mg). After 30 min at room temperature thereaction mixture was partitioned between ethyl acetate and water. Theorganic layer was washed with water and brine, dried and concentrated toa white solid. Trituration with diethyl ether and collection of thesolid afforded the title compound as a white powder (25 mg, 49% yield).TLC(MeOH:CH₂Cl₂, 5:95): R_(f)=0.48.

Example 61N-[(1-Methylindole-2-carbonyl)valinyl]-3-amino-4-oxo-5-fluoropentanoicAcid

A solution ofN-[(1-methylindole-2-carbonyl)valinyl]-3-amino-4-oxo-5-fluoropentanoicacid, t-butyl ester (19 mg, 0.041 mmol) and anisole (0.1 mL) in CH₂Cl₂(1 mL) was treated with trifluoroacetic acid (0.5 mL) at roomtemperature. After 30 min the reaction mixture was concentrated andchased with methylene chloride. The crude residue was triturated withdiethyl ether and the solid filtered to afford the title compound as alight brown solid (12 mg, 72% yield). TLC(AcOH:MeOH:CH₂Cl₂, 1:1:20):R_(f)=0.59. Mass Spectrum for C₂₀H₂₄FN₃O₅: [MH]⁺406, [MH]⁻404.

Following the methods set down in Examples 59-61, the followingcompounds were prepared:

Example 62N-[(1,3-Dimethyl-5-fluoroindole-2-carbonyl)valinyl]-3-amino-4-oxo-5-fluoropentanoicAcid

57% yield; TLC(MeOH:CH₂Cl₂, 5:95): R_(f)=0.56. Mass Spectrum forC₂₁H₂₅F₂N₃O₅: [MH]⁺438, [MH]⁻436.

Example 63N-[(1-Homoallylindole-2-carbonyl)valinyl]-3-amino-4-oxo-5-fluoropentanoicAcid

29% yield; TLC(MeOH:CH₂Cl₂, 1:9): R_(f)=0.33. Mass Spectrum forC₂₃H₂₈FN₃O₅: [MH]⁺446, [MNa]⁺468, [MH]⁻444.

Example 64N-[(1-Methyl-5-fluoroindole-2-carbonyl)valinyl]-3-amino-4-oxo-5-fluoropentanoicAcid

89% yield; TLC(MeOH:CH₂Cl₂,9:1): R_(f)=0.14. Mass Spectrum forC₂₀H₂₃F₂N₃O₅: [MH]⁺424, [MH]⁻422.

Example 65N-[(1-Methyl-3-isobutylindole-2-carbonyl)valinyl]-3-amino-4-oxo-5-fluoropentanoicAcid

50% yield; TLC(MeOH:CH₂Cl₂, 9:1): R_(f)=0.20. Mass Spectrum forC₂₄H₃₂FN₃O₅: [MH]⁺462, [MH]⁻460.

Example 66N-[(1-Methyl-3-phenethylindole-2-carbonyl)valinyl]-3-amino-4-oxo-5-fluoropentanoicAcid

38% yield; TLC(ethyl acetate: hexanes, 1:1): R_(f)=0.19. Mass Spectrumfor C₂₈H₃₂FN₃O₅: [MH]⁺510, [MH]⁻508.

Example 67N-[(1-Methyl-5-obenzylindole-2-carbonyl)valinyl]-3-amino-4-oxo-5-fluoropentanoicAcid

78% yield; TLC(ethyl acetate: hexanes, 1:1): R_(f)=0.17. Mass Spectrumfor C₂₇H₅₀FN₃O₆: [MH]⁺512, [MH]⁻510.

Example 68N-(1,3-Dimethyl-indole-2-carbonyl)-valinyl-3-amino-5-bromo-4-oxo-pentanoicAcid, t-Butyl Ester

1-Hydroxybenzotriazole hydrate (3.19 g, 20.8 mmol) and1-(3′-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDAC)(5.60 g, 29.2 mmol) were added to a stirred solution ofN-carbobenzyloxycarbonyl valine (5.24 g, 20.8 mmol) in methylenechloride/dimethyl formamide (DMF) (60 ml/30 ml) at 0° C. under nitrogen.After 15 min, aspartic acid a-methyl, β-tert-butyl diester (5.00 g, 20.8mmol) was added as a solid followed by neat 4-methylmorpholine (2.40 ml,21.8 mmol). After stirring at 0° C. for 1 hour and at room temperaturefor 5 hours, the mixture was partitioned between ethyl acetate and 5%KHSO₄ solution. The aqueous solution was back-extracted with ethylacetate and the combined extracts were washed with saturated NaHCO₃ andbrine, dried over sodium sulfate, and concentrated to give a solid.Trituration with ether afforded of N-[carbobenzyloxycarbonylvalinyl]aspartic acid, α-methyl, β-tert-butyl diester as a white solid(8.36 g, 92%). TLC(CH₂Cl₂/MeOH, 95/5): R_(f)=0.48.

A solution of the above product (4.00 g, 9.17 mmol) in 200 ml ofmethanol was stirred with palladium on activated carbon (0.45 g) underan atomosphere of hydrogen (1 atm) for 50 min. The reaction mixture wasthen filtered through a pad of Celite and the filter cake was washedwith methanol and methylene chloride. The filtrates were combined andconcentrated, and the residue was chased with methylene chloride to giveN-[valinyl]aspartic acid, α-methyl, β-tert-butyl diester a white solid(2.75 g, 99%). TLC (CH₂Cl₂/MeOH, 95/5): R_(f)=0.10.

To a turbid mixture of the above product (2.75 g, 9.11 mmol) and1,3-dimethylindole-2-carboxylic acid (1.95 g, 10.3 mmol) in DMF (30 ml)was added 4-dimethylaminopyridine (DMAP) (1.26 g, 10.3 mmol) and1-(3′-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDAC)(2.37 g, 12.4 mmol). The reaction mixture was stirred under a nitrogenatmosphere at 0° C. for 1 hour and at room temperature for 3 hours. Thereaction mixture was then partitioned between ethyl acetate and 5% KHSO₄solution and the aqueous solution was back-extracted with ethyl acetate.The combined extracts were washed with saturated NaHCO₃ solution, water,and brine, dried over sodium sulfate, and concentrated to give a solid.The solid was triturated with ether to giveN-[(1,3-dimethylindole-2-carbonyl)valinyl]aspartic acid, α-methyl,β-tert-butyl diester as a white powder (2.87 g, 67%). TLC (CH₂Cl₂/MeOH,95/5): R_(f)=0.59.

An aqueous solution of lithium hydroxide (1.0 M, 2.98 ml) was addeddropwise to a suspension the above product (1.41 g, 2.98 mmol) in1,4-dioxane (10 ml). After stirring at room temperature for 30 min, theresulting clear was acidified with 1 N hydrochloric acid solution anddiluted with water. The resulting white precipitate was collected bysuction filtration and washed successively with water and with a smallamount of ether, affordingN-[(1,3-dimethyl-indole-2-carbonyl)-valinyl]aspartic acid, β-tert-butylester as a white powder (1.18 g, 86%). TLC(CH₂Cl₂/MeOH, 90/10):R_(f)=0.21.

To a solution of the above product (1.03 g, 2.24 mmol) and4-methylmorphorline (0.35 ml, 3.14 mmol) in THF (20 mL) at −10° C. undernitrogen was added dropwise isobutyl chloroformate (0.380 ml, 2.92mmol). The reaction mixture was stirred under nitrogen at −10° C. for 15min and filtered. The filter cake was washed with dry THF and thefiltrates were combined and cooled to 0° C. The filtrates were thentreated with a freshly prepared ether solution of diazomethane (excess).After the mixture was stirred at 0° C. for 1 hour, a mixture ofhydrobromic acid (48% wt. aq. solution) and acetic acid (6 ml, 1/1) wasadded dropwise till the gas evolution ceased. After another 5 min, thereaction mixture was concentrated and partitioned between ethyl acetateand water. The aqueous layer was back-extracted with ethyl acetate. Theorganic layers were combined, washed with water, saturated NaHCO₃solution, and brine, dried over sodium sulfate, and concentrated. Theresidue was triturated with ether to give the title compound as a whitepowder (1.00 g, 83%). TLC(CH₂Cl₂/MeOH, 95/5): R_(f)=0.88.

Example 69N-[(1,3-Dimethyl-indole-2-carbonyl)-valinyl]-3-amino-5-(2,6-dichlorobenzoyl)oxy-4-oxo-pentanoicAcid, t-Butyl Ester

To a mixture of 2,6-dichlorobenzoic acid (0.023 g, 0.12 mmol) andpotassium fluoride (0.015 g, 0.25 mmol) at room temperature undernitrogen was addedN-[(1,3-dimethyl-indole-2-carbonyl)valinyl]-3-amino-5-bromo-4-oxo-pentanoicacid, tert-butyl ester (0.054 g, 0.10 mmol) in one portion. Afterstirring at room temperature for further 16 hrs, the mixture waspartitioned between ethyl acetate and water. The organic layer waswashed with water, saturated NaHCO₃ solution, and brine, dried oversodium sulfate, and concentrated. Trituration with ether gave the titlecompound as a white powder (0.051 g, 79%). TLC(CH₂Cl₂/MeOH, 95/5):R_(f)=0.88.

Example 70N-[N-(1,3-Dimethyl-indole-2-carbonyl)-valinyl]-3-amino-5-(2,6-dichlorobenzoyl)oxy-4-oxo-pentanoicAcid

Trifluoroacetic acid (2 mL) was added to a stirred solution ofN-(1,3-dimethyl-indole-2-carbonyl)-valinyl-3-amino-5-(2,6-dichlorobenzoyl)oxy-4-oxo-pentanoicacid, t-butyl ester (0.0340 g, 0.0526 mmol) in methylene chloridecontaining anisole (0.2 mL). The reaction mixture was stirred at roomtemperature under nitrogen for half an hour and concentrated. Theresidue was azeotroped with methylene chloride and triturated with etherto give the title compound as a white powder (0.0270 g, 87%).TLC(CH₂Cl₂/MeOH/AcOH, 20/1/1): R_(f)=0.43. MS for C₂₈H₂₉Cl₂N₃O₇,[MH]⁺590/592, [MH]⁻588/590.

Following the methods set down in Examples 69-70, the followingcompounds were prepared:

Example 71N-(1,3-Dimethyl-indole-2-carbonyl)-valinyl-3-amino-5-(diphenylphosphinyl)oxy-4-oxo-pentanoicAcid

24% yield; TLC(CH₂Cl₂/MeOH/AcOH, 20/1/1): R_(f)=0.31. MS forC₃₃H₃₆PN₃O₇, [MH]⁺618, [MH]⁻616.

Example 72N-(1,3-Dimethyl-indole-2-carbonyl)-valinyl-3-amino-5-(1-phenyl-3-(trifluoromethyl)pyrazol-5-yl)oxy-4-oxo-pentanoicAcid

49% yield; TLC(CH₂Cl₂/MeOH, 90/10): R_(f)=0.29. MS for C₃₁H₃₂F₃N₅O₆,[MH]⁺628, [MH]⁻626.

Example 73N-(1,3-Dimethyl-indole-2-carbonyl)-valinyl-3-amino-5-(3-(N-phenyl)aminocarbonyl-2-naphthyl)oxy-4-oxo-pentanoicAcid

68% yield; TLC(CH₂Cl₂/MeOH, 80/20): R_(f)=0.46. MS for C₃₈H₃₈N_(4O) ₇,[MH]⁺663, [MH]⁻661.

Example 74N-(1,3-Dimethyl-indole-2-carbonyl)-valinyl-3-amino-5-(2-aminocarbonyl-1-phenyl)oxy-4-oxo-pentanoicAcid

61% yield; TLC(CH₂Cl₂/MeOH/HOAc, 8/1/1): R_(f)=0.32. MS for C₂₈H₃₂N₄O₇,[MH]⁺537, [MH]⁻535.

Example 75N-(1,3-Dimethyl-indole-2-carbonyl)-valinyl-3-amino-5-(dimethylphosphinyl)oxy-4-oxo-pentanoicAcid

76% yield; TLC(CH₂Cl₂/MeOH, 90/10): R_(f)=0.12. MS for C₂₃H₃₂PN₃O₇,[MH]⁺494, [MH]⁻492.

Example 76

(2S-cis)-[5-Benxyloxycarbonylamino-1,2,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)amino]-4-oxo-butanoicAcid tert-Butyl Ester Semicarbazone

1. Preparation of(2S-cis)-5-Benzyloxycarbonylamino-1,2,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carboxylicAcids Ethyl Ester

To a solution of(2S-cis)-5-amino-1,2,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carboxylicacid, ethyl ester (0.437 g, 1.73 mmol, prepared as described inTetrahedron Letters 36, pp. 1593-1596 (1995) and U.S. Pat. No. 5,504,080(Apr. 2, 1996)) in methylene chloride (4 mL) stirring at 0° C. was addedbenzyl chloroformate (0.370 mL, 2.6 mmol) and triethylamine (0.724 mL,5.2 mmol) and the resulting mixture was stirred under nitrogen for 45minutes. The reaction was quenched with water then partitioned betweenethyl acetate and 5% aqueous potassium bisulfate solution. The aqueouslayer was back-extracted two times with ethyl acetate, then the combinedorganic layers were washed with saturated sodium chloride solution,dried over sodium sulfate and evaporated to dryness. Purification of thecrude product by flash chromatography on silica gel (S/P brand silicagel 60 Å, 230-400 mesh ASTM) eluting with ethyl acetate-hexane (2:1)gave 0.558 g (68%) of crude product. Trituration with ethylacetate-hexane (1:4) gave 0.480 g of the title compound as white solid;m.p.: 139-140° C. TLC (ethyl acetate-hexane, 2:1): R_(f)=0.6; ¹H-NMR(300 MHz, CDCl₃): δ 7.35-7.30 (m, 5H), 7.02-6.94 (m, 3H), 6.17 (d, J=5.4Hz, 1H), 4.15 (q, J=7.1 Hz, 2H), 3.46 (dd, J=11.0, 16.7 Hz, 1H), 3.29(m, 1H), 3.10 (d, J=116.5, 2H), 2.35 (m, 1H), 2.16, (m, 1H), 1.23 (t,J=7.2 Hz, 3H).

2. Preparation of(2S-cis)-5-Benzyloxycarbonylamino-1,2,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carboxylicAcid

To a solution of(2S-cis)-5-benzyloxycarbonylamino-1,2,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carboxylicacid, ethyl ester, (0.428 g, 1.05 mmol) in 1,4-dioxane (7.5 mL) andwater (2.5 mL) was added 1M aqueous lithium hydroxide (1.6 mL, 1.6 mmol)and the resulting mixture was stirred at room temperature under nitrogenfor 30 minutes. The reaction mixture was acidified to pH 3 with a 5%aqueous potassium bisulfate sodium chloride solution. The aqueous layerwas back-extracted two times with ethyl acetate, and the combinedorganic layers were dried over sodium sulfate and evaporated to drynessto yield 0.395 g (99%) of title compound as a fine white solid; m.p.:188-189° C. TLC (methylene chloride-methanol-acetic acid, 9:1:1):R_(f)=0.55; ¹H-NMR (300 MHz, CDCl₃) δ 7.34-7.26 (m, 5H), 7.07-6.97 (m,3H), 6.08 (d, J=5.7 Hz, 1H), 5.25 (dd, J=3.2, 9.8 Hz, 1H), 5.10 (s, 2H),4.30 (m, 1H), 3.36 (m, 1H), 3.26 (m, 2H), 3.06 (d, J=12.0 Hz, 1H), 2.36(m, 1H), 2.09 (m, 1H).

3. Preparation ofN-(Benzyloxycarbonyl)-L-(N′-methyl-N′-methoxy)aspartamide β-(tert-ButylEster)

To a solution of N-(benzyloxycarbonyl)-L-asparticacid-β-(tert-butyl)ester (14.65 g, 45.3 mmol, Bachem) in CH₂Cl₂ (150 mL)at 0° C. (ice bath) under a nitrogen atmosphere was added1-hydroxybenzotriazole hydrate (7.29 g, 47.6 mmol, Aldrich) followed by1-ethyl-3-(3′,3′-dimethyl-1′-aminopropyl)carbodiimide hydrochloride(9.55 g, 49.8 mmol, Sigma). After stirring at 0° C. for 15 min.,N,O-dimethylhydroxylamine hydrochloride (5.10 g, 52.3 mmol, Aldrich) andN-methylmorpholine (5.8 mL, 53 mmol, Aldrich) were added. The mixturewas allowed to warm to room temperature over 3 hours then stirred atroom temperature for 16 hours. The solution was concentrated undervacuum and the residue partitioned between ethyl acetate-5% KHSO₄ (200mL each). The organic phase was washed in turn with 5% KHSO₄, saturatedsodium bicarbonate and saturated sodium chloride solutions; dried overanhydrous sodium sulfate and evaporated to an oil. The oil wascrystallized from hexane to give the title product (16.10 g, 97% yield)as a fluffy white crystalline solid. TLC (ethyl acetate), single spot(UV and PMA): R_(f)=0.37.

A similar procedure to the one above, starting with 29.3 g ofN-(benzyloxycarbonyl)-L-aspartic acid-β-(tert-butyl)ester (2-fold scaleup) gave 31.18 g (94% yield) of the title product.

4. Preparation of N-(Benzyloxycarbonyl)-L-aspartic Acid Semicarbazoneβ-(tert-Butyl)Ester

To a solution ofN-(benzyloxycarbonyl)-L-(N′-methyl-N′-methoxy)aspartamide β-(tert-butylester) (15.50 g, 42.3 mmol) in anhydrous ether (400 mL) at 0° C. (icebath) under a nitrogen atmosphere was added dropwise to a 1.0 M solutionof LiAlH₄ in ether (22.0 mL, 22.0 mmol, Aldrich) at such a rate as tokeep the reaction solution temperature between 0-5° C. (addition time15-20 min). After the addition of the lithium aluminum hydride reagentwas complete, the mixture was stirred at 0-5° C. for 1 hr, then quenchedby the dropwise addition of 0.3 N KHSO₄ solution (100 mL). The resultantmixture was transferred to a separatory funnel adding sufficient 5%KHSO₄ solution (75 mL) to dissolve the solids. The organic phase wasseparated and the combined aqueous washes back-extracted with ether (100mL). The combined ether extracts were washed with saturated NaClsolution, dried over anhydrous sodium sulfate and concentrated in vacuowith minimal heating. TLC (ethyl acetate): streaky spot (UV and PMA)R_(f)=0.48. TLC (methanol/methylene chloride, 1:9) major spot (UV andPMA): R_(f)=0.75.

The crude aldehyde was immediately taken up in aqueous ethanol (45 mLwater/105 mL alcohol), placed in an ice bath and treated with sodiumacetate (3.82 g, 46.6 mmol) and semicarbazide hydrochloride (5.20 g,46.6 mmol, Aldrich). The mixture was stirred at 0° C. (ice bath) under anitrogen atmosphere for 3 hrs, allowed to warm to room temperature, andstirred overnight (16 hrs). Most of the ethanol was removed under vacuumand the residue partitioned between ethyl acetate and water (100 mLeach). The organic phase was washed sequentially with 5% KHSO₄,saturated sodium bicarbonate and saturated sodium chloride solutions;dried over anhydrous sodium sulfate and evaporated to dryness. The crudeproduct of this reaction was combined with that of two similarprocedures starting with 15.40 g and 4.625 g ofN-(benzyloxycarbonyl)-L-(N′-methyl-N′-methoxy)aspartamide b-(tert-butylester) (total: 35.525 g, 97 mmol) and these combined products werepurified by flash chromotagraphy on silica gel eluting withacetone/methylene chloride (3:7) then methanol-acetone-methylenechloride (0.5:3:7) to give pure title product (27.73 g, 78.5%) as acolorless foam. TLC (MeOH—CH₂Cl₂, 1:9): single spot (UV and PMA),R_(f)=0.51.

5. Preparation of L-aspartic Acid Semicarbazone β-(tert-Butyl) Ester,p-Toluenesulfonate Salt

5 To a solution of N-(benzyloxycarbonyl)-L-aspartic acid semicarbazoneβ-(tert-butyl)ester (13.84 g, 38.0 mmol) in absolute ethanol (250 mL)was added 10% Pd/C (1.50 g, Aldrich) and the resulting mixture stirredunder an atmosphere of hydrogen (balloon) until TLC (methanol/methylenechloride, 1:9) indicated complete consumption of the starting material(60 min). Note: It is important to follow this reaction closely sincethe product can be over-reduced. The mixture was filtered though Celiteand evaporated to an oil. The oil was chased with methylene chloride(2×75 mL) then with methylene chloride/toluene (1:1, 75 mL) to give thecrude amine as a white crystalline solid. TLC (EtOAc-pyridine-AcOH—H₂O;60:20:5:10) single spot (UV and PMA) R_(f)=0.24. Note: In this TLCsystem, any over-reduced product will show up immediately below thedesired product, R_(f)=0.18 (PMA only).

The crude amine was taken up in CH₃ CN (60 mL) and treated with asolution of p-toluenesulfonic acid monohydrate (7.22 g, 38.0 mmol) inacetonitrile (60 mL). The crystalline precipitate was collected, washedwith acetonitrile and ether, and air-dried to give the title compound(13.95 g, 92% yield) as a white, crystalline solid.

The optical purity of this material was checked by conversion to thecorresponding Mosher amide [1.05 equiv(R)-(−)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride, 2.1equivalents of i-Pr₂ NEt in CH₂ Cl₂, room temperature, 30 min]. Thedesired product has a doublet at 7.13 ppm (1H, d, J=2.4 Hz, CH═N) whilethe corresponding signal for its diastereomer is at 7.07 ppm. Theoptical purity of the title compound obtained from the above procedureis typically>95:5.

6.(2S-cis)-[5-Benzyloxycarbonylamino-1,2,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)amino]-4-oxo-butanoicAcid tert-Butyl Ester Semicarbazone

To a solution of(2S-cis)-5-benzyloxycarbonylamino-1,2,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carboxylicacid (0.375 g, 0.989 mmol) in methylene chloride (7 mL) stirring at 0°C. under nitrogen was added 1-hydroxybenzotriazole hydrate (0.182 g,1.19 mmol) and 1-ethyl-3-(3′,3′-dimethyl- ′-aminopropyl)carbodiimidehydrochloride (0.284 g, 1.48 mmol). After 15 minutes L-aspartic acidsemicarbazone b-(tert-butyl) ester, p-toluenesulfonate salt (0.386 g,0.989 mmol) and N-methylmorpholine (0.163 mL, 1.48 mmol) were added andthe resultant reaction mixture allowed to come to room temperaturewithin 1 hour. After stirring overnight, the reaction mixture wasdiluted with ethyl acetate and washed successively with 5% potassiumbisulfate and saturated sodium chloride solutions; dried over sodiumsulfate and evaporated to dryness. Purification of the crude product byflash chromatography on silica gel (S/P brand silica gel 60 Å, 230-400mesh ASTM) eluting with 2% methanol-methylene chloride gave 0.463 g(79%) of the title compound as a white foam. TLC (methylenechloride-methanol, 9:1): R_(f)=0.5. ¹H-NMR (300 MHz, CDCl₃): δ 8.42(s,1H), 7.82 (d, J=8.1 Hz, 1H), 7.32 (m, 5H), 7.07 (m, 3H), 5.94 (d,J=6.3 Hz, 1H), 5.26 (d, J=9 Hz, 1H), 5.10 (s, 2H), 4.82 (m, 1H, 4.35 (m,1H), 3.56 (d, J=18 Hz, 1H), 3.27 (m, 2H), 3.07 (m, 1H), 2.64 (dd, J=4.7,15.8 Hz, 1H), 2.44 (dd, J=6.6, 15.9 Hz, 2H), 2.22 (m, 1H), 1.30 (s, 9H).Mass spectrum: m/z 593 (M+H).

Example 77

(2-cis)-[5-Benzyloxycarbonylamino-1,2,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicAcid Semicarbazone

To a solution of(2S-cis)-[5-benzyloxycarbonylamino-1,2,4,5,6,7-bexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)amino]-4-oxo-butanoicacid tert-butyl ester semicarbazone (0.214 g, 0.362 mmol) in methylenechloride (1.5 mL) was added anisole (0.5 mL, 4.34 mmol) followed bytrifluoroacetic acid (0.75 mL). After stirring at room temperature undernitrogen for 2 hours the reaction mixture was diluted with methylenechloride and evaporated, then chased twice with methylene chloride togive the title compound (0.195 g). TLC (methylene chloride-methanol,95:5), R_(f)=0.2. ¹H-NMR (300 MHz, CDCl₃) δ 9.77 (bs, 1H), 8.32 (d, J=12Hz, 1H), 8.12 (d, J-7.8 Hz, 1H), 7.31-7.27 (m, 5H) 7.13-7.04 (m, 3H),6.64 (m, 1H) 5.32 (d, J=9.9 Hz, 1H), 5.12 (s, 2H), 4.86 (m, 1H), 4.41(m, 1H), 3.56 (d, J=15 Hz, 1H), 3.25 (m, 2H), 3.10 (m, 2H), 2.64 (m,2H), 2.28 (m, 2H).

Example 78

(2S-cis)-[5-Benzyloxycarbonylamino-1,2,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicAcid

(2-cis)-[5-Benzyloxycarbonylamino-1,2,4,5,6,7-hexadydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicacid semicarbazone (0.195 g, 0.36 mmol) was treated with a 3:1:1solution of methanol-acetic acid-37% formaldehyde (2 mL) and theresulting mixture stirred under nitrogen for 1.5 hours. The reactionmixture was diluted with water, methanol removed by evaporation, thenthe remaining mixture lyophilized. Purification of the crude product byflash chromatography on reverse phase gel (MCI gel, CHP-20P, 75-150micron)eluting with a 10%-80% methanol-water gradient gave 0.073 g,(42%) of the title compound as a white solid after lyophilization; m.p.101-104° C. TLC (methylene chloride-methanol-acetic acid, 97:2.5:0.5)R_(f)=0.45. ¹H-NMR (300 MHz, CDCl₃) δ 7.45 (m, 1H), 7.30 (s, 5H), 7.07(d, J=3.3 Hz, 1H), 7.00 (d, J=4.8 Hz, 2H), 6.12)m, 1H), 5.17 (d, J=9.6Hz, 1H), 5.07 (s, 2H), 4.49 (m, 1H), 4.28 (m, 1H), 3.46 (d, J=9.9 Hz,1H), 3.30-3.12 (m, 2H), 3.04-2.99 (m, 1H), 2.83-2.76 (m, 1H), 2.46-2.33(m, 2H), 2.03 (bs, 1H). Mass spectrum: m/z 480 (M+H).

Example 79

(2S-cis)-[5-amino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicAcid tert-Butyl Ester Semicarbazone

10% Palladium on carbon (0.180 g) was added to a solution of(2S-cis)-[5-benzyloxycarbonylamino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]4-oxo-butanoicacid tert-butyl ester semicarbazone (0.308 g, 0.520 mmol) in methanol(27 mL) and the resulting mixture was hydrogenated using a balloon ofhydrogen (1 atm, R.T.) for 18 hours. The mixture was filtered throughCelite, evaporated to dryness, then chased two times with toluene togive the title compound as an off-white solid (0.215 g). TLC, (methylenechloride-methanol, 9:1) R_(f)=0.15. ¹H-NMR (300 MHz, CDCl₃) δ 8.53 (s,1H), 7.89 (d, J=7.6 Hz, 1H), 7.13 (m, 3H), 5.21 (dd, J=2.3, 10.14 Hz,1H), 4.82 (m, 1H), 3.52 (m, 1H), 3.24 (dd, J=10.3, 16.3 Hz, 1H), 3.03(m, 2H), 2.62 & 2.42 (AB, dd, J=4.2, 7.1, 15.7 Hz, 2H), 2.19 (m, 1H),1.32 (s, 9H).

Example 80

(2S-cis)-[5-(N-Acetyl-(S)-aspartyl-b-tert-butylester)amino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicAcid tert-Butyl Ester Semicarbazone

To a solution of N-acetyl aspartic acid, β-tert-butyl ester (0.120 g,0.517 mmol) in methylene chloride (1.5 mL) stirring at 0° C. undernitrogen was added 1-hydroxybenzotriazole hydrate (0.086 g, 0.564 mmol)and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.135g, 0.705 mmol). After 15 minutes, a solution of(2S-cis)-[5-amino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicacid tert-butyl ester semicarbazone (0.213 g, 0.47 mmol) in methylenechloride (2 mL) was added and the reaction was allowed to come to roomtemperature over 1 hour. After stirring overnight, the reaction mixturewas diluted with ethyl acetate and washed successively with 5% potassiumbisulfate and saturated sodium chloride solutions; dried (sodiumsulfate) and evaporated to dryness. Purification of the crude product byflash chromatography on silica gel (S/P brand silica gel 60 Å, 230-400mesh ASTM) eluting with 5% then 10% methanol-methylene chloride gave0.126 g (41%) of the title compound as a white solid. TLC (methylenechloride-methanol, 9:1) R_(f)=0.4. ¹H-NMR (300 MHz, CDCl₃) δ 9.63 (s,1H), 8.32 (d, J=7.8 Hz, 1H), 7.82 (d, J=6.6 Hz, 1H), 7.53 (d, J=4.8 Hz,1H), 7.09 (m, 1H), 7.00 (m, 2H), 5.18 (d, J=8.1 Hz, 1H), 4.86 (m, 1H),4.39 (m, 1H), 3.01 (m, 1H), 2.92 (dd, J=4.2, 14.7 Hz, 1H) 2.68 (d,J=12.3 Hz, 1H), 2.52 (m, 2H), 2.51 (m, 2H), 2.03 (s, 3H), 1.39 (s, 9H),1.24 (s, 9H).

Example 81

(2S-cis)-[5-(N-Acetyl-(S)-aspartyl)amino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicAcid Semicarbazone

To a solution of (2S-cis)-[5-(N-acetyl-(S)-aspartyl-b-tert-butylester)amino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbony1)-amino]-4-oxo-butanoic acid tert-butyl ester semicarbazone (0.117 g,0.178 mmol) in methylene chloride (1 mL) was added anisole (0.5 mL)followed by trifluoroacetic acid (1 mL). After stirring at roomtemperature under nitrogen for 2 hours, the reaction mixture was dilutedwith methylene chloride and evaporated, then chased twice with methylenechloride to give the title compound (0.099 g). TLC (methylenechloride-methanol-acetic acid, 13:6:1) R_(f)=0.2. Mass spectrum: m/z 560(M+H).

Example 82

(2S-cis)-[5-(N-Acetyl-(S)-aspartyl)amino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicAcid

(2S-cis)-[5-(N-Acetyl-(S)-aspartyl)amino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicacid semicarbazone (0.097 g, 0.177 mmol), was treated with a 3:1:1solution of methanol-acetic acid-37% formaldehyde (2 mL) and theresulting mixture stirred under nitrogen for 1.5 hours. The reactionmixture was then diluted with water, methanol removed by evaporation,then the remaining mixture lyophilized. Purification of the crudeproduct by flash chromatography on reverse phase gel (MCI gel, CHP-20P,75-150 micron) eluting with a 10%-80% methanol-water gradient gave 0.050g (56%) of the title compound as a white solid after lyophilization;m.p. 160-175° C. (dec). TLC (methylene chloride-methanol-acetic acid,13:6:1) R_(f)=0.3. Mass spectrum: m/z 503 (M+H).

Example 83

(2S-cis)-[5-Succinylamino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicAcid tert-Butyl Ester Semicarbazone

To a solution of(2S-cis)-[5-amino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicacid tert-butyl ester semicarbazone (0.197 g, 0.435 mmol) in methylenechloride (6 mL) stirring at 0° C. under nitrogen was added succinicanhydride (0.057 g, 0.566 mmol), followed by pyridine (0.052 mL, 0.653mmol). After stirring at room temperature under nitrogen for 3 hours,the reaction mixture was diluted with ethyl acetate and washedsuccessively with 5% potassium bisulfate and saturated sodium chloridesolutions; dried (sodium sulfate) and evaporated to dryness.Purification of the crude product by flash chromatography on silica gel(S/P brand silica gel 60 Å, 230-400 mesh ASTM) eluting with 10%methanol-methylene chloride then 80:19:1 methylenechloride-methanol-acetic acid gave 0.216 g (88%) of the title compoundas a white solid. TLC (methylene chloride-methanol-acetic acid, 8:1:1)R_(f)=0.5. Mass spectrum: m/z 557 (M−H).

Example 84

(2S-cis)-[5-Succinylamino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicAcid Semicarbazone

To a solution of(2S-cis)-[5-succinylamino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]4-oxo-butanoicacid tert-butyl ester semicarbazone (0.191 g, 0.342 mmol) in methylenechloride (1 mL) was added anisole (0.5 mL) followed by trifluoroaceticacid (1 mL). After stirring at room temperature under nitrogen for 2hours, the reaction mixture was diluted with methylene chloride andevaporated, then chased twice with methylene chloride to give the titlecompound (0.210 g). TLC (methylene chloride-methanol-acetic acid, 8:1:1)R_(f)=0.4. Mass spectrum: m/z 503 (M+H).

Example 85

(2S-cis)-[5-Succinylamino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicAcid

(2S-cis)-[5-Succinylamino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicacid semicarbazone (0.208 g, ca. 0.342 mmol), was treated with a 3:1:1solution of methanol-acetic acid-37% formaldehyde (3 mL), and theresulting mixture stirred under nitrogen for 1.5 hours. The reactionmixture was then diluted with water, methanol removed by evaporation,then the remaining mixture lyophilized. Purification of the crudeproduct by flash chromatography on reverse phase gel (MCI gel, CHP-20P,75-150 micron) eluting with a 10%-80% methanol-water gradient gave 0.064g (42%) of the title compound as a white solid after lyophilization;m.p. 145-160° C. (dec). TLC (methylene chloride-methanol-acetic acid,8:1:1) R_(f)=0.45. Mass spectrum: m/z 446.

Example 86

(2S-cis)-[5-(N-Benzyloxycarbonyl-(S)-aspartyl-b-tert-butylester)amino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicAcid tert-Butyl Ester Semicarbazone

To a solution of N-benzyloxycarbonyl-(S)-aspartyl-p-tert-butyl ester(0.169 g, 0.521 mmol) in methylene chloride (1.5 mL) stirring at 0° C.under nitrogen was added 1-hydroxybenzotriazole hydrate (0.087 g, 0.569mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride(0.136 g, 0.711 mmol). After 15 minutes, a solution of(2S-cis)-[5-amino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicacid tert-butyl ester semicarbazone (0.217 g, 0.474 mmol) in methylenechloride (2 mL) was added and the reaction was allowed to come to roomtemperature within 1 hour. After stirring overnight, the reactionmixture was diluted with ethyl acetate and washed successively with 5%potassium bisulfate and saturated sodium chloride solutions; dried(sodium sulfate) and evaporated to dryness. Purification of the crudeproduct by flash chromatography on silica gel (S/P brand silica gel 60Å, 230-400 mesh ASTM) eluting with 2% then 5% methanol-methylenechloride gave 0.244 g (67%) of the title compound as an off-white solid.TLC (methylene chloride-methanol, 9:1) R_(f)=0.55. ¹H-NMR (300 MHz,CDCl₃) δ 9.13 (s, 1H), 7.85 (d, J=6 Hz, 1H), 7.56 (d, J=5.7 Hz, 1H) 7.23(m, 5H), 7.08 (m, 1H), 7.00 (m, 2H), 5.13 (m, 3H) 4.77 (m, 1H), 4.62 (m,1H), 4.43 (m, 1H), 3.60 (d, J=16 Hz, 1H), 3.22 (m, 2H), 2.98 (m, 1H),2.83 (d, J=15.3 Hz, 1H), 2.65 & 2.36 (AB, dd, J=4.2, 7.7, 16.9 Hz, 2H),2.42 (m, 1H), 2.10 (m, 1H), 1.35 (s, 9H), 1.24 (s, 9H).

Example 87

(2S-cis)-[5-(N-Benzyloxycarbonyl-(S)-aspartyl)amino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicAcid Semicarbazone

To a solution of(2S-cis)-[5-(N-Benzyloxycarbonyl-(S)-aspartyl-b-tert-butylester)amino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicacid tert-butyl ester semicarbazone (0.217 g, 0.289 mmol) in methylenechloride (1 mL) was added anisole (0.5 mL) followed by trifluoroaceticacid (1 mL). After stirring at room temperature under nitrogen for 3hours, the reaction mixture was diluted with methylene chloride andevaporated, then chased twice with methylene chloride to give the titlecompound (0.193 g). TLC (methylene chloride-methanol, 9:1) R_(f)=0.35.Mass spectrum: m/z 652 (M+H).

Example 88

(2S-cis)-[5-(N-Benzyloxycarbonyl-(S)-aspartyl)amino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicAcid

(2S-cis)-[5-(N-Benzyloxycarbonyl-(S)-aspartyl)amino-1,2,3,4,5,6,7-hexahydro4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicacid semicarbazone (0.191 g, 0.29 mmol), was treated with a 3:1:1solution of methanol-acetic acid-37% formaldehyde (2 mL) and theresulting mixture stirred under nitrogen for 2 hours. The reactionmixture was then diluted with water, methanol removed by evaporation,then the remaining mixture lyophilized. Purification of the crudeproduct by flash chromatography on reverse phase gel (MCI gel, CHP-20P,75-150 micron) eluting with a 10%-80% methanol-water gradient gave 0.111g. (64%) of the title compound as a white solid after lyophilization;m.p. 140-144° C. (dec.). TLC (methylene chloride-methanol, 9:1)R_(f)=0.4. Mass spectrum: m/z 593 (M−H).

Example 89

(2S-cis)-[5-Dihydrocinnamylamino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicAcid tert-Butyl Ester Semicarbazone

To a solution of dihydrocinnamic acid (0.169 g, 0.521 mmol) in methylenechloride (1.5 mL) stirring at 0° C. under nitrogen was added1-hydroxybenzotriazole hydrate (0.088 g, 0.576 mmol) and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.127 g,0.665 mmol). After 15 minutes, a solution of(2S-cis)-[5-amino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicacid tert-butyl ester semicarbazone (0.203 g, 0.443 mmol) in methylenechloride (2 mL), was added and the reaction was allowed to come to roomtemperature within 1 hour. After stirring overnight, the reactionmixture was diluted with ethyl acetate and washed successively with 5%potassium bisulfate and saturated sodium chloride solutions; dried(sodium sulfate) and evaporated to dryness. Purification of the crudeproduct by flash chromatography on silica gel (S/P brand silica gel 60Å, 230-400 mesh ASTM) eluting with 2 then 5% methanol-methylene chloridegave 0.208 g (79%) of the title compound as an off-white solid. TLC(methylene chloride-methanol, 9:1) R_(f)=0.7. ¹H-NMR (300 MHz, CDCl₃) δ8.82 (s, 1H), 7.72 (d, J=8.1 Hz, 1H), 7.19 (m, 5H), 7.06 (m, 1H), 7.01(m, 2H), 6.76 (d, J=6.3, 1H, 5.23 (d, J=8.4 Hz, 1H0, 4.84 (m, 1H), 4.50(m, 1H), 3.48 (m, 1H), 3.26 (m, 2H), 3.05 (m, 1H), 2.94 (m, 2H), 2.53(m, 4H), 2.28 (m, 1H), 2.06 (m, 1H), 1.29 (s, 9H). Mass spectrum: m/z591 (M+H).

Example 90

(2S-cis)-[5-Dihydrocinnamylamino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicAcid Semicarbazone

To a solution of(2S-cis)-[5-dihydrocinnamylamino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicacid tert-butyl ester semicarbazone (0.189 g, 0.320 mmol) in methylenechloride (1 mL) was added anisole (0.5 mL) followed by trifluoroaceticacid (1 mL). After stirring at room temperature under nitrogen for 3hours, the reaction mixture was diluted with methylene chloride andevaporated, then chased twice with methylene chloride to give the titlecompound (0.183 g). TLC(methylene chloride-methanol, 9:1) R_(f)=0.25.Mass spectrum: m/z 535 (M+H).

Example 91

(2S-cis)-[5-Dihydrocinnamylamino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicAcid

(2S-cis)-[5-Dihydrocinnamylamino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicacid semicarbazone (0.181 g, ca. 0.320 mmol), was treated with a 3:1:1solution of methanol-acetic acid-37% formaldehyde (2 mL) and theresulting mixture stirred under nitrogen for 4 hours. The reactionmixture was then diluted with water, methanol removed by evaporation,then the remaining mixture lyophilized. Purification of the crudeproduct by flash chromatography on reverse phase gel (MCI gel, CHP-20P,75-150 micron) eluting with a 10%-80% methanol-water gradient gave 0.075g (47%) of the title compound as a white solid after lyophilization;m.p. 78-81° C. TLC (methylene chloride-methanol, 9:1) R_(f)=0.45. ¹H-NMR(300 MHz, DMSO d6): δ 8.58 (m, 1H), 8.30 (d, J=7.5 Hz, 1H), 7.24 (m,5H), 7.08 (m, 2H), 6.99 (m, 1H), 5.04 (d, J=9.3 Hz, 1H), 4.39 (m, 1H),4.19 (m, 1H), 3.46 (m, 1H), 3.05 (m, 2H), 2.93 (d, J=16.8 Hz, 2H), 2.83(m, 2H), 2.00 (d, J=5.1 Hz, 2H). Mass spectrum: m/z 478 (M+H).

Example 92

(2S-cis)-[5-Acetylamino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicAcid tert-Butyl Ester Semicarbazone

To a solution of(2S-cis)-[5-amino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicacid tert-butyl ester semicarbazone (0.222 g, 0.490 mmol) in pyridine (3mL) at room temperature under nitrogen was added acetic anhydride (0.07mL, 0.735 mmol). After stirring overnight, the reaction mixture wasdiluted with methylene chloride and evaporated to give a foam. This wastaken up in ethyl acetate and washed successively with 5% potassiumbisulfate and saturated sodium chloride solutions; dried (sodiumsulfate) and evaporated to dryness to give 0.130 g (53%) of the titlecompound as an off-white solid. TLC (methylene chloride-methanol, 9:1)R_(f)=0.55. ¹H-NMR (300 MHz, CDCl₃): δ 8.75 (s, 1H), 7.75 (d, J=8.4 Hz,1H), 7.08 (m, 1H), 7.01 (m, 2H), 6.87 (d, J=6.3 Hz, 1H), 5.25 (d, J=8.1Hz, 1H), 4.84 (m, 1H), 4.52 (m, 1H), 3.50 (m, 1H), 3.28 (m, 2H), 3.02(m, 1H), 2.55 & 2.46 (AB, dd, J=4.2, 7.1, 15.7 Hz, 2H), 2.36 (m, 1H),2.18 (m, 1H), 2.02 (s, 3H), 1.31 (s, 9H).

Example 93

(2S-cis)-[5-Acetylamino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicAcid Semicarbazone

To a solution of(2S-cis)-[5-acetylamino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicacid tert-butyl ester semicarbazone (0.112 g, 0.224 mmol) in methylenechloride (1 mL) was added anisole (0.5 mL) followed by trifluoroaceticacid (1 mL). After stirring at room temperature under nitrogen for 2.5hours, the reaction mixture was diluted with methylene chloride andevaporated, then chased twice with methylene chloride to give the titlecompound (0.117 g). TLC (methylene chloride-methanol, 9:1) R_(f)=0.15.Mass spectrum: m/z 445 (M+H).

Example 94

(2S-cis)-[5-Acetylamino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicAcid

(2S-cis)-[5-Acetylamino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1hi]indole-2-carbonyl)-amino]-4-oxo-butanoicacid semicarbazone (0.115 g, ca. 0.224 mmol) was treated with a 3:1:1solution of methanol-acetic acid-3 7% formaldehyde (2 mL) and theresulting mixture stirred under nitrogen for 5 hours. The reactionmixture was diluted with water, methanol removed by evaporation, thenthe remaining mixture lyophilized. Purification of the crude product byflash chromatography on reverse phase gel (MCI gel, CHP-20P, 75-150micron) eluting with a 10%-80% methanol-water gradient gave 0.044 g(51%) of the title compound as a white solid after lyophilization; m.p.210-215° C. (dec). TLC (methylene chloride-methanol-acetic acid, 44:5:1)R_(f)=0.45. Mass spectrum: m/z 388 (M+H).

Example 95

(2S-cis)-[5-(1-Naphthoyl)amino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino]3,2,1-hi]indole-2-carbonyl)-amino-4-oxo-butanoic Acid tert-Butyl Ester Semicarbazone

To a solution of 1-naphthoic acid (0.072 g, 0.417 mmol) in methylenechloride (1.5 mL) stirring at 0° C. under nitrogen was added1-hydroxybenzotriazole hydrate (0.077 g, 0.501 mmol.) and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.120 g,0.626 mmol). After 15 minutes, a solution of(2S-cis)-[5-amino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicacid tert-butyl ester semicarbazone (0.189 g, 0.147 mmol) in methylenechloride (2 mL), was added and the reaction was allowed to come to roomtemperature within 1 hour. After stirring a total of 5 hours, thereaction mixture was diluted with ethyl acetate and washed successivelywith 5% potassium bisulfate and saturated sodium chloride solutions,dried (sodium sulfate) and evaporated to dryness. Purification of thecrude product by flash chromatography on silica gel (S/P brand silicagel 60 Å, 230-400 mesh ASTM) eluting with 5% methanol-methylene chloridegave 0.168 g (66%) of the title compound as an off-white solid; m.p.103-105° C. (dec.). TLC (methylene chloride-methanol, 9:1) R_(f)=0.6.Mass spectrum: m/z 613 (M+H). ¹H-NMR (300 MHz, CDCl₃) Δ 9.09 (bs, 1H),8.38 (d, J=8.4 Hz, 1H), 7.82-7.93 (m, 3H), 7.70 (d, J=6.3 Hz, 1H),7.45-7.58 (m, 3H), 7.37 (d, J=6.6 Hz, 1H), 7.06-7.15 (m, 4H), 5.30 (d,J=8.4 Hz, 1H), 4.80-4.85 (m, 2H), 3.57 (d, J=3.6 Hz, 1H), 3.30-3.45 (m,2H), 3.16 (m, 1H), 2.59-2.65 (m, 2H), 2.27-2.49 (m, 2H), 1.29 (s, 9H).

Example 96

(2S-cis)-[5-(1-Naphthoyl)amino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicAcid Semicarbazone

To a solution of(2S-cis)-[5-(1-naphthoyl)amino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicacid tert-butyl ester semicarbazone (0.106 g, 0.173 mmol) in methylenechloride (1 mL) was added anisole (0.5 mL) followed by trifluoroaceticacid (1 mL). After stirring at room temperature under nitrogen for 3hours, the reaction mixture was diluted with methylene chloride andevaporated, then chased twice with methylene chloride to give the titlecompound (0.110 g). TLC (methylene chloride-methanol, 9:1) R_(f)=0.3.

Example 97

(2S-cis)-[5-(1-Naphthoyl)amino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicAcid

(2S-cis)-[5-(1-Naphthoyl)amino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoic acid semicarbazone (0.110g, ca. 0.173 mmol) was treated with a 3:1:1 solution of methanol-aceticacid-37% formaldehyde (3 mL) and the resulting mixture stirred undernitrogen for 5 hours. The reaction mixture was then diluted with water,methanol removed by evaporation, then the remaining mixture lyophilized.Purification of the crude product by flash chromatography on silica gel(S/P brand silica gel 60 Å, 230-400 mesh ASTM) eluting with a 5%-20%methanol-methylene chloride gradient gave 0.076 g (86%) of the titlecompound as a white solid; m.p. 202-203° C. (dec). TLC(methylenechloride-methanol-acetic acid, 20:1:1) R_(f)=0.3. Mass spectrum: m/z 498(M−H). ¹H-NMR (300 MHz, CDCl₃) Δ 9.38 (bs, 1H), 8.94 (m, 1H), 8.56 (m,1H), 8.36 (m, 1H), 7.94-8.02 (m, 2H), 7.68 (d, J=6.9 Hz, 1H), 7.51-7.59(m, 3H), 7.07-7.13 (m, 2H), 6.97 (m, 1H), 5.20 (d, J=10.5, 1 Hz), 4.67(m, 1H), 4.15 (m, 1H), 3.49 (m, 1H), 2.95-3.23 (m, 2H), 2.53 (m, 1H),2.22-2.34 (m, 2H).

Example 98

(2S-cis)-[5-Benzoylamino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicAcid tert-Butyl Ester Semicarbazone

To a solution of(2S-cis)-[5-amino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicacid tert-butyl ester semicarbazone (0.121 g, 0.264 mmol) in methylenechloride (2.5 mL) stirring at 0° C. under nitrogen was addedtriethylamine (0.055 mL, 0.396 mmol), followed by benzoyl chloride(0.037 mL, 0.317 mmol). After stirring at room temperature undernitrogen for 1 hour, the reaction mixture was diluted with ethyl acetateand washed successively with 5% potassium bisulfate, saturated sodiumbicarbonate and saturated sodium chloride solutions; dried (sodiumsulfate) and evaporated to dryness. Purification of the crude product byflash chromatography on silica gel (S/P brand silica gel 60 Å, 230-400mesh ASTM) eluting with 10% hexane-ethyl acetate, 100% ethyl acetate,then 10% methanol-ethyl acetate gave 0.073 g (49%) of the title compoundas a off-white solid. TLC (methylene chloride-methanol, 9:1) R_(f)=0.7.Mass spectrum: m/z 563 (M+H). ¹H-NMR (300 MHz, CDCl₃): Δ 8.61 (bs, 1H),7.83-7.86 (m, 2H), 7.47-7.53 (m, 3H), 7.06-7.12 (m, 3H), 5.30 (dd,J=2.2, 7.8 Hz, 1H), 4.89 (m, 1H), 4.72 (m, 1H), 3.60 (d, J=16.5 Hz, 1H),3.36 (m, H), 3.19 (m, 1H), 2.69 (dd, J=4.4, 11.7 Hz, 1H), 2.52 (m, 1H),2.29 (m, 1H), 1.34 (s, 9H).

Example 99

(2S-cis)-[5-Benzoylamino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicAcid Semicarbazone

To a solution of(2S-cis)-[5-benzoylamino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicacid tert-butyl ester semicarbazone (0.064 g, 0.114 mmol) in methylenechloride (1 mL) was added anisole (0.5 mL) followed by trifluoroaceticacid (1 mL). After stirring at room temperature under nitrogen for 2.5hours, the reaction mixture was diluted with ethyl acetate andevaporated to give the title compound (0.070 g). TLC (methylenechloride-methanol, 4:1) R_(f)=0.4. Mass spectrum: m/z 507 (M+H).

Example 100

(2S-cis)-[5-Benzoylamino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicAcid

(2S-cis)-[5-Benzoylamino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]-4-oxo-butanoicacid semicarbazone (0.070 g, ca. 0.114 mmol) was treated with a 3:1:1solution of methanol-acetic acid-37% formaldehyde (3 mL), and theresulting mixture stirred under nitrogen for 3.5 hours. The reactionmixture was then diluted with water, methanol removed by evaporation,then the remaining mixture lyophilized. Purification of the crudeproduct by flash chromatography on silica gel (S/P brand silica gel 60Å, 230-400 mesh ASTM) eluting with 10 and 20% methanol-methylenechloride gave 0.042 g (82%) of the title compound as a white solid; m.p.204-205° C. (dec). TLC (methylene chloride-methanol, 4:1) R_(f)=0.4.Mass spectrum: m/z 448 (M−H). ¹H-NMR (300 MHz, DMSO-d₆) Δ 8.84 (m, 1H),8.53 (m, 1H), 7.91-7.95 (m, 2H), 7.46-7.58 (m, 3H), 7.11 (m, 2H), 6.99(t, J=7.3 Hz, 1H), 5.14 (d, 10.2 Hz, 1H), 4.62 (m, 1H), 4.23 (m, 1H),3.48 (m, 1H), 3.12-3.18 (m, 2H), 2.99 (m, 1H), 2.58 (m, 1H), 2.12-2.46(m, 3H).

Example 101

(3R,S-cis)-6-Benzyloxycarbonylamino-5-oxo-2,3,4,5,6,7,8-hexahydro-1H-azepino[3,2,1-hi]quinoline-3-carbonyl)-amino]-4-oxo-butanoicAcid tert-Butyl Ester Semicarbazone

1. Preparation of(3R,S-cis)-6-Benzyloxycarbonylamino-5-oxo-2,3,4,5,6,7,8-hexahydro-1H-azepino[3,2,1-hi]quinoline-3-carboxylicAcid, Methyl Ester

To a solution of(3R,S-cis)-6-Amino-5-oxo-2,3,4,5,6,7,8-hexahydro-1H-azepino[3,2,1-hi]quinoline-3-carboxylicacid, methyl ester (0.570 g, 2.1 mmol, prepared as described inTetrahderon Letters 36, pp. 1593-1596 (1995) and U.S. Pat. No. 5,504,080(Apr. 2, 1996)) in methylene chloride (6 mL) stirring at 0° C. was addedbenzyl chloroformate (0.6 mL, 4.2 mmol) and triethylamine (1.2 mL, 8.4mmol) and the resulting mixture was stirred under nitrogen for 30minutes. The reaction was quenched with water then partitioned betweenethyl acetate and 5% aqueous potassium bisulfate solution. The aqueouslayer was back extracted two times with ethyl acetate, then the combinedorganic layers were washed with saturated sodium chloride solution,dried (sodium sulfate) and evaporated to dryness. Purification of thecrude product by flash chromatography on silica gel (S/P brand silicagel 60 Å, 230-400 mesh ASTM) eluting with ethyl acetate-hexane (2:1)gave 0.643 g (76%) of the title compound as a white foam. TLC (methylenechloride-methanol, 95:5) R_(f)=0.8. ¹H-NMR (300 MHz, CDCl₃) Δ 7.36-7.25(m, 5H), 7.13-7.02 (m, 3H), 5.67 (d, J=7.8 Hz, 1H), 5.02 (t, J=9.15,18.3 Hz, 2H), 4.34 (m, 1H), 3.70 (s, 3H), 3.16 (m, 1H), 2.69-2.56 (m,5H), 2.06 (m, 1H). Mass spectrum: m/z 408 (M+H).

2. Preparation of(3R,S-cis)-6-Benzyloxycarbonylamino-5-oxo-2,3,4,5,6,7,8-hexahydro-1H-azepino[3,2,1-hi]quinoline-3-carboxylicAcid

To a solution of(3R,S-cis)-6-Benzyloxycarbonylamino-5-oxo-2,3,4,5,6,7,8-hexahydro-1H-azepino[3,2,1-hi]quinoline-3-carboxylicacid, methyl ester (0.622 g, 1.53 mmol) in 1,4-dioxane (10.5 mL) andwater (3.5 mL) was added 1M aqueous lithium hydroxide (2.3 mL, 2.3 mmol)and the resulting mixture was stirred at room temperature under nitrogenfor 1 hour. The reaction mixture was acidified to ca. pH 2 with a 5%aqueous potassium bisulfate solution, then partitioned between ethylacetate and saturated sodium chloride solution. The aqueous layer wasback extracted two times with ethyl acetate, and the combined organiclayers were dried (sodium sulfate) and evaporated to yield 0.670 g ofthe title compound. TLC (methylene chloride-methanol-acetic acid,32:1:1) R_(f)=0.35. ¹H-NMR (300 MHz, CDCl₃): Δ 7.38-7.28 (m, 5H),7.13-7.04 (m, 3H), 5.72 (d, J=8.1 Hz, 1H), 5.03 (s, 2H), 4.35 (m, 1H),3.77-3.67(m, 5H), 3.10 (m, 1H), 2.72-2.52 (m, 5H), 2.07 (m, 1H), 1.70(m, 1H).

3.(3R,S-cis)-6-Benzyloxycarbonylamino-5-oxo-2,3,4,5,6,7,8-hexahydro-1H-azepino[3,2,1-hi]quinoline-3-carbonyl)-amino]-4-oxo-butanoicAcid tert-Butyl Ester Semicarbazone

To a solution of(3R,S-cis)-6-benzyloxycarbonylamino-5-oxo-2,3,4,5,6,7,8-hexahydro-1H-azepino[3,2,1-hi]quinoline-3-carboxylicacid (0.604 g, 1.5 mmol) in methylene chloride (12 mL) stirring at 0° C.under nitrogen was added 1-hydroxybenzotriazole hydrate (0.282 g, 1.8mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride(0.442 g, 3 mmol). After 15 minutes, L-aspartic acid semicarbazoneβ-tert-butyl ester, p-toluenesulfonate salt (0.60 g, 1.5 mmol) andN-methylmorpholine (0.25 mL, 3 mmol) were added and the mixture allowedto come to room temperature within 1 hour. After stirring an additionalhour, the reaction mixture was diluted with ethyl acetate and washedsuccessively with 5% potassium bisulfate and saturated sodium chloridesolutions; dried (sodium sulfate) and evaporated to dryness.Purification of the crude product by flash chromatography on silica gel(S/P brand silica gel 60 Å, 230-400 mesh ASTM) eluting with 10%methanol-methylene chloride gave 0.523 g (56%) of the title compound asa white foam. TLC (methylene chloride-methanol, 9:1) R_(f)'0.65. ¹H-NMR(300 MHz, CDCl₃): Δ 9.89 (m, 1H), 7.72 (m, 1H), 7.92 (d, J=9 Hz, 1H),7.65 (d, J=8.1 Hz, 1H), 7.32-7.28 (m, 5H), 7.12 (s, 1H), 7.07 (d, J=5.7Hz, 2H), 603 (d, J=7.5 Hz, 1H), 5.84 (d, J=8.1 Hz, 1H), 5.03 (s, 2H),5.01 (m, 1H) 4.80 (m, 1H), 4.31 (m, 1H), 2.98 (m, 1H), 2.75-2.41 (m,7H), 2.12 (m, 1H), 1.77 (m, 1H), 1.39 (s, 9H).

Example 102

(3R,S-cis)-6-Benzyloxycarbonylamino-5-oxo-2,3,4,5,6,7,8-hexahydro-1H-azepino[3,2,1-hi]quinoline-3-carbonyl)-amino]-4-oxo-butanoicAcid Semicarbazone

To a solution of(3R,S-cis)-6-Benzyloxycarbonylamino-5-oxo-2,3,4,5,6,7,8-hexahydro-1H-azepino[3,2,1-hi]quinoline-3-carbonyl)-amino]-4-oxo-butanoicacid tert-butyl ester semicarbazone (0.200 g, 0.33 mmol) in methylenechloride (1 mL) was added anisole (0.5 mL, 4.62 mmol) followed bytrifluoroacetic acid (1 mL). After stirring at room temperature undernitrogen for 1.5 hours the reaction mixture was diluted with methylenechloride and evaporated, then azeotroped twice with methylene chlorideto give the title compound (0.248 g). TLC (methylenechloride-methanol-acetic acid, 8:1:1) R_(f)=0.2. Mass spectrum: m/z 549[M−H].

Example 103

(3R,S-cis)-6-Benzyloxycarbonylamino-5-oxo-2,3,4,5,6,7,8-hexahydro-1H-azepino[3,2,1-hi]quinoline-3-carbonyl)-amino]-4-oxo-butanoicAcid

(3 R,S-cis)-6-Benzyloxycarbonylamino-5-oxo-2,3,4,5,6,7,8-hexahydro-1H-azepino[3,2,1-hi]quinoline-3-carbonyl)-amino]-4-oxo-butanoicacid semicarbazone (0.245 g, ca 0.33 mmol), was treated with a 3:1:1solution of methanol-acetic acid-37% formaldehyde (3 mL) and theresulting mixture stirred under nitrogen for 1.5 hours. The reactionmixture was diluted with water, methanol removed by evaporation, thenthe remaining mixture lyophilized. Purification of the crude product byflash chromatography on reverse phase gel (MCI gel, CHP-20P, 75-150micron) eluting with a 10%-80% methanol-water gradient gave 0.090 g(60%) of the title compound as a white solid after lyophilization; m.p.120-123° C. (dec). TLC (methylene chloride-methanol-acetic acid, 32:1:1)R_(f)=0.45. ¹H-NMR (300 MHz, DMSO d6): Δ 8.67 (m, 1H), 7.79 (m, 1H),7.57 (m, 1H), 7.37-7.27 (m, 5H), 7.17-7.08 (m, 3H), 5.44 (m, 1H), 4.95(s, 2H), 4.70 (m, 1H), 4.07 (m, 1H), 3.92 (m, 1H), 3.16 (m, 1H), 2.98(m, 1H), 2.75-2.41 (m, 7H), 2.25 (m, 1H), 2.11(m, 1H), 1.29 (m, 1H).Mass spectrum: m/z 492 [M−H].

Example 104

3{(2S-cis)-[5-Benzyloxycarbonylamino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]}-5-fluoro-4-hydroxy-pentanoicAcid tert-Butyl Ester

To a solution of(2S-cis)-5-benzyloxycarbonylamino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carboxylicacid (0.373 g, 0.98 mmol) in methylene chloride (3 mL) sting at 0° C.under nitrogen was added 1-hydroxybenzotriazole hydrate (0.151 g, 0.98mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride(0.283 g, 1.47 mmol). After 15 minutes,3-amino-4-hydroxy-5-fluoropentanoic acid, tert-butyl ester (0.204 g,0.98 mmol, prepared as described in Tetrahedron Letters 35, pp.9693-9696 (1994)) was added and the mixture allowed to come to roomtemperature within 1 hour. After stirring overnight, the reactionmixture was diluted with ethyl acetate and washed successively with 5%potassium bisulfate and saturated sodium chloride solutions; dried(sodium sulfate) and evaporated to dryness. Purification of the crudeproduct by flash chromatography on silica gel (S/P brand silica gel 60Å, 230-400 mesh ASTM) eluting with 2% methanol-methylene chloride gave0.383 g (68%) of the title compound as a white foam. TLC (methylenechloride-methanol, 9:1) R_(f)=0.6. ¹H-NMR (300 MHz, CDCl₃): Δ7.45-7.31(m, 5H), 7.08-7.01 (m, 3H), 6.10 (m, 1H), 5.26 (m, 1H), 5.12(s, 2H), 4.52 (m, 1H), 4.38-4.30 (m, 2H), 4.21-4.19 (m, 2H), 4.03-3.95(m, 2H), 3.43-3.20 (m, 4H), 3.13 (m, 2H), 2.62-2.50 (m, 2H), 2.42 (m,1H), 1.42 (s, 4H), 1.32 (s, 5H). Mass spectrum: m/z 570 (M+H).

Example 105

3{(2S-cis)-[5-Benzyloxycarbonylamino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]}-5-fluoro-4-oxo-pentanoicAcid tert-Butyl Ester

To a solution of3{(2S-cis)-[5-benzyloxycarbonylamino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]}-5-fluoro-4-hydroxy-pentanoicacid tert-butyl ester (0.114 g, 0.20 mmol) in methyl sulfoxide (1.3 mL)was added Dess-Martin periodinane (0.228 g). After stirring at roomtemperature under nitrogen for 2 hours an additional portion ofDess-Martin periodinane (0.135 g) was added followed 2.5 hours later bya third portion (0.10 g). The reaction mixture was diluted with ethylacetate and washed twice with water and saturated sodium chloridesolution; dried (sodium sulfate) and evaporated to dryness. Purificationof the crude product by flash chromatography on silica gel (S/P brandsilica gel 60 Å, 230-400 mesh ASTM) eluting 1/1 ethyl acetate-hexanesgave 0.076 g (67%) of the title compound as a white foam. TLC (ethylacetate-hexanes, 1:1) R_(f)=0.6. ¹H-NMR (300 MHz, CDCl₃): Δ 7.58 (d,J=8.4 Hz, 1H), 7.34-7.30 (m, 5H), 7.07-6.99 (m, 3H), 6.06 (m, 1H), 5.23(d, J=12.3 Hz, 1H), 5.12 (s, 2H), 4.53 (d, J=13.2 Hz, 1H), 4.77 (d,J=9.9 Hz, 2H), 4.32 (m, 1H), 3.44 (dd, J=5, 8.4 Hz, 1H), 3.32-3.21 (m,2H), 3.06 (m, 1H), 2.9 (m, 1H), 2.62 (m, 1H), 2.41 (m, 1H), 2.17 (m,1H), 1.39 (s, 4H), 1.29 (s, 5H).

Example 106

3{(2S-cis)-[5-Benzyloxycarbonylamino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]}-5-fluoro-4-oxo-pentanoicAcid

To a solution of3{(2S-cis)-[5-benzyloxycarbonylamino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl)-amino]}-5-fluoro-4-oxo-pentanoic acidtert-butyl ester (0.063 g, 0.111 mmol) in methylene chloride (1.0 mL)was added anisole (0.5 mL), followed by trifluoroacetic acid (1.0 mL).After stirring at room temperature under nitrogen for 2 hours thereaction mixture was diluted with methylene chloride and evaporated,then chased twice with methylene chloride. The crude residue wastriturated with ethyl ether to give 0.030 g of the titled product as awhite solid; m.p. 106-107° C. (dec). TLC (methylenechloride-methanol-acetic acid, 32:1:1) R_(f)=0.3. ¹H-NMR (300 MHz,CDCl₃): Δ 7.61 (m, 1H), 7.32 (s, 5H), 7.1(d, J=4 Hz, 1H), 7.03 (d, J=4Hz, 2H), 6.17 (m, 1H), 5.22 (m, 1H), 5.10 (s, 2H), 4.75-4.70 (m, 2H),4.32 (m, 1H), 3.5 (m, 1H), 3.31-3.15 (m, 2H), 3.03 (m, 1H), 2.93 (m,1H), 2.69 (m, 1H), 2.36 (m, 1H), 2.12 (m, 1H). Mass spectrum: m/z 512(M+H).

Example 107

3-[(2S-cis)-[5-Benzyloxycarbonylamino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl]amino]-5-bromo-4-oxo-pentanoicAcid, tert-Butyl Ester

To a solution of(2S-cis)-5-benzyloxycarbonylamino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carboxylicacid (0.302 g, 0.797 mmol) in methylene chloride (5.5 mL) stirring at0C. under nitrogen was added 1-hydroxybenzotriazole hydrate (0.146 g,0.96 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (0.230 g, 1.2 mmol). After 15 minutes, aspartic acid,α-methyl, β-tert-butyl diester hydrochloride (0.191 g, 0.797 mmol) wasadded followed by N-methylmorpholine (0.13 mL, 1.2 mmol) and the mixtureallowed to come to room temperature within 1 hour. After stirringovernight, the reaction mixture was diluted with ethyl acetate-andwashed successively with 5% potassium bisulfate and saturated sodiumchloride solutions; dried (sodium sulfate) and evaporated to dryness.Purification of the crude product by flash chromatography on silica gel(S/P brand silica gel 60 Å, 230-400 mesh ASTM) eluting with ethylacetate-hexane (1:1) gave 0.350 g (78%) ofN-[(2S-cis)-[5-benzyloxy-carbonylamino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl]]asparticacid, α-methyl, β-tert-butyl diester as a white solid. TLC (methylenechloride-methanol, 9:1) R_(f)=0.8. m.p. 147-148° C.(dec.). ¹H-NMR(300MHz, CDCl₃): Δ 7.48 (d, J=7.5 Hz, 1H), 7.34-7.29 (m, 5H), 7.07 (m, 1H),7.03-6.96 (m, 2H), 6.15 (d, J=5.7 Hz, 1H), 5.28 (d, J=7.8 Hz 1H), 5.11(s, 2H), 4.72 (m, 1H), 4.32 (m, 1H), 3.74 (s, 3H), 3.49 (d, J=16.5 Hz,1H), 3.31-3.20 (m, 2H), 3.05 (m, 1H), 2.72 (ABX, dd, J=4.65, 15, 64.5Hz, 2H), 2.43 (m, 1H), 2.15 (m, 1H), 1.30 (s, 9H).

To a solution of the above product (0.330 g, 0.585 mmol) in 1,4-dioxane(4.5 mL) and water (1.5 mL) was added IM aqueous lithium hydroxide (0.7mL, 0.702 mmol) and the resulting mixture was stirred at roomtemperature under nitrogen for 30 minutes. The reaction mixture wasacidified to pH 3 with a 0.1N HCl solution, then partitioned betweenethyl acetate and saturated sodium chloride solution. The aqueous layerwas back extracted two times with ethyl acetate, and the combinedorganic layers were dried (sodium sulfate) and evaporated to yield 0.275g (85%) ofN-[(2S-cis)-[5-benzyloxycarbonylamino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl]]asparticacid, β-tert-butyl ester as a white foam. TLC(methylenechloride-methanol, 9:1): R_(f)=0.25. ¹H-NMR(300 MHz, CDCl₃): d 7.57 (d,J=7.8 Hz, 1H), d 7.35-7.29 (m, 5H), 7.08 (m, 1H), 7.03-6.98 (m, 2H),6.24 (d, J=6 Hz, 1H), 5.28 (d, J=5.1 Hz 1H), 5.11 (s, 2H), 4.73 (m, 1H),4.35 (m, 1H), 3.48 (d, J=16.8 Hz, 1H), 3.36-3.20 (m, 2H), 3.07 (m, 1H),2.76 (ABX, dd, J=4.8, 18, 66 Hz, 2H), 2.40 (m, 1H), 2.19 (m, 1H), 1.33(s, 9H).

To a solution of the above product (0.262 g, 0.475 mmol) intetrahydrofuran (3.0 mL) stirring at −10° C. under nitrogen was addedN-methylmorpholine (0.114 mL, 1.05 mmol) followed by dropwise additionof isobutyl chloroformate (0.107 mL, 0.81 mmol). After 40 minutes thereaction mixture was filtered, the salts washed with dry THF, and thefiltrate cooled to 0° C. This was treated with a freshly preparedethereal solution of diazomethane (excess). After stirring the mixtureat 0° C. for 30 minutes, a mixture of hydrobromic acid (48% wt. aq.solution)/acetic acid (1.3 mL, 1/1) was added dropwise. After stirringfor another 10 minutes, the reaction mixture was diluted with ethylacetate, then washed successively with saturated sodium bicarbonate andsaturated sodium chloride solutions; dried (sodium sulfate) andevaporated to dryness. Purification of the crude product by flashchromatography on silica gel (S/P brand silica gel 60 Å, 230-400 meshASTM) eluting with ethyl acetate-hexane (1:1) gave 0.200 g (67%) of thetitle compound as a white foam. TLC(ethyl acetate-hexane, 1:1):R_(f)=0.7. ¹H-NMR (300 MHz, CDCl₃): Δ 7.71 (d, J=9 Hz, 1H), d7.35-7.30(m, 5H), 7.09 (m, 1H), 7.04-7.02 (m, 2H), 6.1(d, J=5.4 Hz, 1H), 5.28 (d,J=7.2 Hz 1H), 5.12 (s, 2H), 4.89 (dd, J=4.5, 15 Hz 1H), 4.35 (m, 1H),4.16 (s, 2H), 3.50-3.21(m, 3H), 3.06 (m, 1H), 2.76 (ABX, dd, J=4.65, 18,103 Hz, 2H), 2.37 (m, 1H), 2.15 (m, 1H), 1.27 (s, 9H). Mass spectrum:m/z 626/628 (M−H).

Example 108

3-[(2S-cis)-[5-Benzyloxycarbonylamino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl]amino]-5-(diphenylphosphinyl)oxy-4-oxo-pentanoicAcid, tert-Butyl Ester

To a solution of3-[(2S-cis)-[5-benzyloxycarbonylamino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl]amino]-5-bromo-4-oxo-pentanoic acid,tert-butyl ester (0.069 g, 0.110 mmol) in N,N-dimethylformamide (1.0 mL)was added potassium fluoride (0.029 g, 0.495 mmol), followeddiphenyiphosphinic acid (0.029 g, 0.139 mmol). After stirring at roomtemperature under nitrogen for 48 hours, the reaction mixture wasdiluted with ethyl acetate, then washed successively with a dilutesodium bicarbonate solution then water; dried (sodium sulfate) andevaporated to dryness. Purification of the crude product by flashchromatography on silica gel (S/P brand silica gel 60 Å, 230-400 meshASTM) eluting with ethyl acetate-hexane (1:1) gave 0.048 g (59%) of thetitle compound as a clear oil. TLC(ethyl acetate-hexane, 2:1):R_(f)=0.3. ¹H-NMR(300 MHz, CDCl₃): Δ 7.89-7.80 (m, 4H), 7.52-7.30 (m,11H), 7.06 (m, 1H), 7.01-6.96 (m, 2H), 6.45 (m, 1H), 5.21 (m, 1H), 5.13(s, 2H), 4.96 (dd, J=8.3, 18 Hz, 1H), 4.78-4.70 (m, 2H), 4.35 (m, 1H),3.35-3.23 (m, 3H), 3.05 (m, 1H), 2.76 (ABX, dd, J=4.65, 18, 103 Hz, 2H),2.43(m, 1H), 2.18 (m, 1H), 1.33 (s, 9H).

Example 109

3-[(2S-cis)-[5-Benzyloxycarbonylamino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl]amino]-5-(diphenylphosphinyl)oxy-4-oxo-pentanoicAcid

To a solution of3-[(2S-cis)-[5-benzyloxycarbonylamino-1,2,3,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carbonyl]amino]-5-(diphenylphosphinyl)oxy-4-oxo-pentanoicacid, tert-butyl ester (0.040 g, 0.054 mmol) in methylene chloride (1.0mL) was added anisole (0.5 mL), followed by trifluoroacetic acid (1.0mL). After stirring at room temperature under nitrogen for 30 minutesthe reaction mixture was diluted with methylene chloride and evaporated,then azeotroped twice with methylene chloride. The crude residue wastriturated with ethyl ether to give 0.030 g of the titled product as awhite solid; m.p. 109-111° C. (dec). TLC(methylene chloride-methanol,9:1): R_(f)=0.4. ¹H-NMR(300 Mhz, CDCl₃): Δ 7.87-7.66 (m, 4H),7.60-7.28.(m, 11H), 7.05-6.95 (m, 3H), 6.84 (m, 1H), 5.12(s, 2H), 5.05(m, 1H), 4.58 (m, 1H), 4.42-4.15 (m, 4H), 3.35-3.10 (m, 4H), 3.05 (m,1H), 2.76 (m, 1H), 2.56 (m, 1H), 2.37(m, 1H), 2.13 (m, 1H), 1.93 (bs,1H). Mass spectrum: m/z 710 (M+H).

Example 110 Materials and Methods for Evaluating Effects of ICE/CED-3Inhibitors on Granulocyte Neutrophils

Neutrophil Isolation:

Whole blood anticoagulated with Acid Citrate Dextrose (ACD) with a ratioof 1:5 ACD to blood was collected (˜100 ml).

Using polypropylene plastic ware, neutrophils are isolated as follows:

30 ml of the whole blood is added to 50 ml polypropylene centrifugetubes containing 15 ml of 6% Dextran (in Saline). The blood is allowedto sediment for approximately 1 hour at room temperature.

The turbid straw colored layer harvested from the top of the cylindersinto 50 ml conical polypropylene tubes. The blood cells were pelleted bycentrifugation at 240×g (Sorvall centrifuge at 1200 rpm) for 12 min. at4° C. with the brake on low.

The supernatant was aspirated and the pooled pellet resuspended in 40-50ml cold PBS (w/o Ca, Mg), and centrifuged at 240×g (Sorvall centrifugeat 1200 rpm) for 6 min. at 4° C. with the brake on high.

The supernatant was aspirated and the pellet resuspended in 12 ml ofcold cell culture grade water. The suspension was titriated gently witha pipet for 30 seconds then add 4 ml of cold 0.6 M KCl. (Brought up to50 ml with cold PBS (w/o Ca, Mg)) and then centrifuged at 300×g (Sorvallcentrifuge at 1400 rpm) for 6 min. at 4° C. with the brake on high.

The above was repeated one time.

The supernatant was aspirated and the cells resuspended in 2.5 ml coldPBS (w/o Ca, Mg). The cell suspension was layered over 3 mlFicoll-Hypaque in a 15 ml polypropylene conical tube and centrifuged at400×g (Sorvall centrifuge at 1900 rpm) for 30 min. at 4° C. with thebrake on low.

The suspension aspirated was down to the neutrophil pellet. The pelletwas resuspended in cold PBS (w/o Ca, Mg) and transfered to a 50 mlconical tube and brought to 50 ml with cold PBS (w/o Ca, Mg) andcentrifuged at 300×g (Sorvall centrifuge at 1400 rpm) for 6 min. at 4°C. with the brake on high.

The supernatant was aspirated and the pellet resuspended in 50 ml coldPBS (w/o Ca, Mg) and centrifuged at 300×g (Sorvall centrifuge at 1400rpm) for 6 min. at 4° C. with the brake on high.

The supernatant was aspirated and the neutrophil pellet resuspended in4.0 ml cold PBS (w/o Ca, Mg) on ice. 10 μl of the neutrophil cellsuspension was diluted with 990 μl of Trypan blue (1:100) and cellscounted using a hemacytometer. The cell number and viability weredetermined.

Neutrophil Culture Conditions:

The culture media was as follows: (RPMI 1640; 10% FBS; 10 mM Hepes; 0.2mM L-glutamine; 25 U/ml penicillin; and 25 mg/ml streptomycin).

Purified neutrophil maintenance was performed under the followingconditions: (5×10⁶ cells/ml in above culture media; Polystyreneround-bottom 96-well plates; 250 μl/well; and 37° C., 5% CO₂/95% airhumidified incubator) (Liles et al., Blood 119 (1995) 3181-3188).

Analysis of Hypodiploid Nuclei by Flow Cytometry:

Hypotonic fluorochrome solution (50 μg /ml propidium iodide (Sigmacatalog #P4170); 0.1% Triton X-100; and 0.1% sodium citrate).

Neutrophils were pelleted at 4° C. and the supernate aspirated.

Neutrophils were resuspended in hypotonic fluorochrome solution at adensity of 5×106 cells/ml. Propidium iodide fluorescence of individualnuclei was evaluated in FL2 and measured on a logarithmic scale whilegating on physical parameters in forward and side scatter to exlude celldebris.

At least 10,000 events per sample were collected and the results wereevaluated relative to a non-apoptotic neutrophil population. (Liles etal., Blood 119 (1995) 3181-3188).

Respiratory burst in isolated neutrophils measured by Chemiluminescence.

Whole blood anticoagulated with Acid Citrate Dextrose (ACD) with a ratioof 1:5 ACD to blood was collected (150 ml).

Neutrophils were isolated as described above.

Opsonized zymosan was prepared by suspending 125 mg zymosan particles in25 ml pooled human serum (5 mg/ml) and incubating them for 20 minutes at37° C. Centrifuge the suspension and resuspend the particles in 7 ml ofPBS (18 mg/ml) and stored on ice until use (vortex prior to pipetting).

50 ml of a 250 μM solution of Lucigenin (MW 510.5) was prepared bydissolving 6.4 mg of the solid in 50 ml of PBS-G (+Ca, Mg). 10 μl ofPBS-G (+Ca, Mg) to the wells in a white 96 well plate.

50 μl of the 250 μM Lucigenin solution was added to the wells in a white96 well plate.

Cell preparations were obatined from cell culture (concentration at timezero=5.0×10⁶ cells/ml) with PBS-G (+Ca, Mg).

20 μl of the neutrophil suspension was suspended to the appropriatewells and the plate was incubated the plate at 37° C. for three minutes.10 μl of the opsonized zymosan was added to the wells.

The plate was read on the luminometer (Labsystems Luminoskan, NeedhamHeights, Mass.) for 14 min. at 37° C. in the kinetic mode and recordresults using the software DeltaSoft.

Whole Blood Assay:

The following reagents were used:

anti-CD32-FITC monoclonal antibody obtained from Pharmingen.

Lysing Solution (10×Stock: 89.9 g NH4Cl;

10.0 g KHCO;

0.37 g tetrasodium EDTA;

dissolve in 1 liter dH2O. Adjust to pH 7.3. Store at 4° C. in a tightlyclosed bottle.

Dilute 1:10 with dH2O prior to use.)

(DPBS without calcium or magnesium obtained from Irivine Scientific.

2% fetal bovine serum in DPBS stored at 4° C.

50 μg/ml propidium iodide in DPBS sterile filtered and stored at 4° C.)

The following protocol was followed:

200 μl blood sample/2.8 ml 1×lysis solution in a 15 ml polypropyleneconical tube.

Cap and invert to mix. Leave at room temperature for 3-5 minutes.

Centrifuge in a table-top Sorvall at 1200 rpm for 5 minutes at 4° C.

Aspirate supernate. Resuspend pellet in 200 μl/sample 2% FBS/DPBS. Add20 μl/sample anti-CD32-FITC. Incubate 30 minutes on ice in the dark.

Add 5 ml/sample DPBS. Centrifuge at 1000 rpm for 5 minutes at 4° C.

Aspirate supernate. Resuspend pellet in 1 ml/sample 2% FBS/DPBS.

Add 3 ml/sample ice-cold 95% EtOH dropwise while vortexing gently.

Incubate samples on ice in the dark for 30 minutes.

Centrifuge at 1000 rpm for 5 minutes at 4° C.

Resuspend each sample in 50 μl 5 mg/ml RN'ase. Transfer sample to 900μl/sample 50 μg/ml Propidium Iodide in 12×75 mm Falcon polystyrenetubes.

Incubate on ice for 30 minutes.

Analyze samples by flow cytometry (argon laser) for forward and sidescatter and fluorescence.

Example 111 Enhancement of Neutrophil/Granulocyte Survival by Ex-VivoApplication of ICE/CED-3 Inhibitors

The present invention provides methods to enhance the ex vivo survivalof neutrophils/granulocytes. To establish the ability of compounds topreserve granulocytes in culture, compounds were tested in a number ofin vitro assays. One common model to test for effects on granulocytesurvival involves separating granulocytes from fresh whole blood,culturing the cells at 37° C. and testing cells for nuclear hypodiploidyat 24 hour intervals (as described in Example 110). The presence ofhypodiploid DNA is a measure of apoptosis, and is assessed using apropidium iodide stain via flow cytometry. Compounds of the presentinvention were incubated with the granulocytes in culture, their effectson granulocyte survival measured, and an IC50 calculated. In FIG. 6, thecaspase inhibitor zVADfmk prepared as described in Tetrahedron Letter,35 9693-9696 (1994) had a weak effect on improving granulocyte survivalat 48 hours, whereas examples 43, 70, and 106 from the present inventionhad IC50s of <5 μM and thus are potent inhibitors of granulocyte death.

The ability to undergo the respiratory burst is another measure ofgranulocyte viability. The respiratory burst is a physiological responseof granulocytes to foreign stimuli such as bacteria. In this example,the method for inducing the respiratory burst utilized opsonizedbacterial zymosan. The respiratory burst was measured viachemiluminescence. FIG. 7 shows that the caspase inhibitor zVADfmk,which had only weak effects on the viability of the granulocytes in thehypodiploidy experiments, did not maintain the respiratory burst. Incontrast, two exemplary compounds of the present invention, example 43and 70, substantially maintained the respiratory burst for 48 hours, andpartially maintained the respiratory burst after granulocyte culture for72 hours.

Survival of granulocytes in whole blood was measured by hypodiploidanalysis in a similar fashion to isolated granulocytes via flowcytometry. ICE/ced-3 inhibitors of the present invention maintainedsurvival of granulocytes in whole blood for 96 hours at room temperatureas indicated in the table below:

Percentage of diploid granulocytes in whole blood time zero 96 hours nocompound 96% 48% EXAMPLE 43 96% 91% EXAMPLE 70 96% 89%

Thus, the present invention provides methods for maintaining the ex vivosurvival of mature granulocytes, both isolated and in whole blood. Themethods of this example also provide a means to distinguish thoseICE/ced-3 inhibitors that are effective in maintaining granulocytesurvival from those that are not effective.

Example 112 Enhancement of Apheresis Product Survival by Application ofICE/CED-3 Inhibitors

Apheresis (leukapheresis) of blood donors can be performed to obtain apopulation of cells which is enriched in granulocytes. These cells arethen transfused into a recipient in need of additional granulocytes.This apheresis product has a short shelf life, and current standards(American Association of Blood Banks, Standard for Blood Banks andTransfusion Services, Ed. 17, 1996) require storage at 20-24° C. for nolonger than 24 hours. Transfusion is recommended within 6 hours ifpossible.

Exemplary compounds as described in the present invention can be used toprolong the storage life of apheresis products. ICE/ced-3 inhibitors areeffective in prolonging granulocyte survival as shown in Example 64 forisolated granulocytes and whole blood. For use in the setting ofapheresis, the compound can be formulated in a compatible solvent, suchas dimethyl sulfoxide (DMSO). The compound can be stored in a vial, andbe pre-added to the apheresis bag, or injected into the donor apheresisline during the collection process. The effective final concentrationcompound could range from 1-25 μM. The leukapheresis product, containingthe ICE/ced-3 inhibitor, is then infused into the recipient afterstorage. Many storage conditions may be possible, for example, storagemay be at room temperature for up to one week post-collection.

Example 113 Bone Marrow Reconstitution/Hematopoietic Cell Recovery

In order to demonstrate utility in accelerating reconstitution ofhematopoietic cells, compounds are tested in several animal models. Onecommon model to destroy hematopoietic cells involves lethal orsub-lethal irradiation of mice, in which the animals are exposed toX-rays, usually generated from a ¹³⁷Cesium source. In the case ofsub-lethal irradiation, various endpoints measuring hematopoiesis can bedetermined at different times following the irradiation, in order tomonitor recovery. Compounds of the present invention are administered toanimals following sublethal irradiation and their effects on recoveryare measured. The endpoints can include: hematocrit, white blood cellcount, incorporation of tritiated thymidine into bone marrow DNA, spleenweight, number of burst-forming units-erythroid or number ofcolony-forming units (erythroid, granulocyte/macrophage andmegakaryocyte forming lineages) from spleen or bone marrow from humerusor femur. In some cases animals can be further myelosuppressed byinjection of chemotherapeutic agents, such as carboplatinum. Compoundswhich have been efficacious in these models, although acting viadifferent mechanisms, include thrombopoeitin, Photofrin, heme,interleukin-1 and tetrachlorodecaoxide, among others.

Models of lethal irradiation can also be used to test efficacy, althoughthese models generally include bone marrow transplant to rescue theanimals. These experiments are often performed on mice, although notexclusively. For syngeneic transplants, time to engraftment can then bemeasured, using the same endpoints as mentioned above. Engraftment ofhuman progenitor cells can also be measured using SCID-hu mice as therecipients with methods similar to the above, after first establishingthe human origin of the hematopoietic cells. In these cases, compoundsof the present invention are administered to recipient animals at thetime of bone marrow transplant. The bone marrow cells may also betreated with the ICE/CED-3 family inhibitor compounds prior totransplantation.

Example 114 Transplantation

Transplantation studies have been performed in a variety of species witha number of organ systems, including heart, liver and kidney. The goalsof these studies have included methods to optimize tissue storage priorto transplantation, and methods to minimize graft rejection. Thisapplication will focus on optimizing tissue storage, althoughapplications to prolong graft function and minimize rejection are alsopossible.

Cardioplegic solutions designed to paralyze the heart are used forcardiopulmonary bypass and for transplantation. The most common of thesesolutions are the University of Wisconsin solution and St. Thomas'Hospital solution. The duration of time the heart can be stored prior totransplant is limited to several hours. Various experimental approacheshave been taken to test additives to these solutions to prolong cardiacviability. The heart can be removed from the donor animal and functionstudied in an isolated preparation (commonly known as a Langendorffpreparation) (Galinanes et al., J. Thoracic & Cardiovascular Surgery104:151, 1992). Decline in cardiac function can be measured as afunction of time in storage, and the parameters measured can includecardiac output, left ventricular pressure, contractile force, heart rateand coronary flow.

The effects of preservation solutions have also been studied for liverand kidney transplants. In this example, the organs are generally storedfor a certain time in the solution, and then grafted into the recipient.Common endpoints for these studies are length of survival andhistological examination of the graft. For liver transplants, thefunction of the graft can also be evaluated by measuring concentrationsof alanine and aspartate aminotransferase in blood. For kidneytransplants, graft function can be assessed by measuring bloodconcentrations of urea, blood urea nitrogen (BUN), and creatine kinase.

As mentioned above, proper storage of organs for transplant is criticalto maintain organ viability. Given the shortage of available organs, anyincrease in the amount of time organs can be maintained, particularlyfrom cadavers, would be of major importance to transplantation medicine.The heart seems to be particularly susceptible to damage duringisolation and storage. Improvement in these conditions could lead to anincrease in the number of viable hearts available for transplant. Animproved organ storage solution can also be used to perfuse the organprior to removal, possibly additionally enhancing its protective effect.Methods of the present invention provide such an improved method,consisting of adding ICE/CED-3 inhibitors to the storage solutions.

Example 115 Inflammation and Analgesia Model

To test anti-inflammatories, an acceptable model is the carrageenan footedema test. Performed with materials, reagents and proceduresessentially as described by Winter, et al., (Proc. Soc. Exp. Biol. Med.111:544 (1962), Male Sprague-Dawley rats are selected in each group sothat the average body weight is as close as possible. Rats are fastedwith free access to water for over sixteen hours prior to the test. Therats are dosed orally (1 mL) with test compounds suspended in vehiclecontaining 0.5% methylcellulose and 0.025% surfactant, or with vehiclealone. One hour later a subplantar injection of 0.1 mL of 1% solution ofcarrageenan/sterile 0.9% saline is administered and the volume of theinjected foot is measured with a displacement plethysmometer connectedto a pressure transducer with a digital indicator. Three hours after theinjection of the carrageenan, the volume of the foot is again measured.The average foot swelling in a group of drug-treated animals is comparedwith that of a group of placebo-treated animals and the percentageinhibition of edema is determined (Otterness and Bliven, LaboratoryModels for Testing NSAIDs, in Non-Steroidal Anti-Inflammatory Drugs, (J.Lombardino, ed. 1985)).

The analgesia test using rat carrageenan is performed with materials,reagents and procedures essentially as described by Hargreaves, et al.,(Pain 32:77 (1988)). Male Sprague-Dawley rats are treated as previouslydescribed, for the Carrageenan Foot Pad Edema test. Three hours afterthe injection of the carrageenan, the rats are placed in a specialplexiglass container with a transparent floor having a high intensitylamp as a radiant heat source, positionable under the floor. After aninitial twenty minute period, thermal stimulation was begun on eitherthe injected foot or on the contralateral uninjected foot. Aphotoelectric cell turned off the lamp and timer when light isinterrupted by paw withdrawal. The time until the rat withdraws its footis then measured. The withdrawal latency in seconds is determined forthe control and drug-treated groups, and percent inhibition of thehyperalgesic foot withdrawal determined.

Although the invention has been described with reference to the examplesprovided above, it should be understood that various modifications canbe made without departing from the spirit of the invention. Accordingly,the invention is limited only by the claims.

We claim:
 1. A method for preventing or treating inflammation, comprising contacting a cell population with an inhibiting effective amount of a reagent that suppresses the protease activity of at least one member of the interleukin-1 beta-converting enzyme (ICE)/CED-3 family, thereby preventing or treating inflammation.
 2. The method of claim 1, wherein said inflammation is chronic inflammation.
 3. The method of claim 1, wherein said inflammation is acute inflammation.
 4. The method of claim 1, wherein said inflammation is due to an inflammatory disease.
 5. The method of claim 4, wherein said inflammatory disease is selected from the group consisting of septic shock, septicemia, and adult respiratory distress syndrome.
 6. The method of claim 1, wherein the reagent suppresses the protease activity in an irreversible manner.
 7. The method of claim 1, wherein the reagent suppresses the protease activity in a reversible manner.
 8. The method of claim 1, wherein the reagent is a compound of formula 1:

wherein: n is 1 or 2; R¹ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, (substituted)phenyl, phenylalkyl, (substituted)phenylalkyl, heteroaryl, (heteroaryl)alkyl or (CH₂)_(m)CO₂R⁴, wherein m=1-4, and R⁴ is as defined below; R² is a hydrogen atom, chloro, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, (substituted)phenyl, phenylalkyl, (substituted)phenylalkyl, heteroaryl, (heteroaryl)alkyl or (CH₂)_(p)CO₂R⁵, wherein p=0-4, and R⁵ is as defined below; R³ is a hydrogen atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted)phenylalkyl; R⁴ is a hydrogen atom alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted)phenylalkyl; R⁵ is a hydrogen atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted)phenylalkyl; A is a natural and unnatural amino acid; B is a hydrogen atom, a deuterium atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, (substituted)phenyl, phenylalkyl, (substituted)phenylalkyl, heteroaryl, (heteroaryl)alkyl, halomethyl, CH₂ZR⁶, CH₂OCO(aryl), CH₂OCO(heteroaryl); or CH₂OPO(R⁷)R⁸ where Z is an oxygen or a sulfur atom; R⁶ is phenyl, substituted phenyl, phenylalkyl, substituted phenylalkyl, heteroaryl, or (heteroaryl)alkyl; and R⁷ and R⁸ are independently selected from a group consisting of alkyl, cycloalkyl, phenyl, substituted phenyl, phenylalkyl, (substituted phenyl) alkyl, and (cycloalkyl) alkyl; and X and Y are independently selected from the group consisting of a hydrogen atom, halo, trihalomethyl, amino, protected amino, an amino salt, mono-substituted amino, di-substituted amino, carboxy, protected carboxy, a carboxylate salt, hydroxy, protected hydroxy, a salt of a hydroxy group, lower alkoxy, lower alkylthio, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, (cycloalkyl)alkyl, substituted (cycloalkyl)alkyl, phenyl, substituted phenyl, phenylalkyl, and (substituted phenyl)alkyl; or a pharmaceutically acceptable salt thereof.
 9. The method of claim 1, wherein the reagent is a compound of formula 3:

wherein: n is 1 or 2; m is 1 or 2; A is R²CO—, R³—O—CO—, or R⁴SO₂—; a group of the formula:

further wherein: R¹ is a hydrogen atom, alkyl or phenylalkyl; R² is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; R³ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted phenyl)alkyl; R⁴ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; R⁵ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; R⁶ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted phenyl)alkyl; R⁷ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; R⁸ is an amino acid side chain chosen from the group consisting of natural and unnatural amino acids; B is a hydrogen atom, a deuterium atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, (heteroaryl)alkyl, or halomethyl; a group of the formula: —CH₂XR⁹; wherein R⁹ is phenyl, substituted phenyl, phenylalkyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; and X is an oxygen or a sulfur atom; a group of the formula: —CH₂—O—CO-(ARYL); a group of the formula: —CH₂—O—CO-(HETEROARYL); a group of the formula: —CH₂—O—PO(R¹⁰)R¹¹ wherein R¹⁰ and R¹¹ are independently selected from a group consisting of alkyl, cycloalkyl, phenyl, substituted phenyl, phenylalkyl and (substituted phenyl) alkyl; and the pharmaceutically-acceptable salts thereof.
 10. A composition comprising a cosmetic, a reagent that suppresses the protease activity of at least one member of the interleukin-1 beta-converting enzyme (ICE)/CED-3 family and a cosmetically or dermatologically acceptable carrier, adapted for preventing or ameliorating irritation of the skin of a mammal due to said cosmetic.
 11. The composition of claim 10, wherein the reagent suppresses the protease activity in an irreversible manner.
 12. The composition of claim 10, wherein the reagent suppresses the protease activity in a reversible manner.
 13. The composition of claim 10, wherein the reagent is a compound of formula 1:

wherein: n is 1 or 2; R¹ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, (substituted)phenyl, phenylalkyl, (substituted)phenylalkyl, heteroaryl, (heteroaryl)alkyl or (CH₂)_(m)CO₂R⁴ wherein m=1-4, and R⁴ is as defined below; R² is a hydrogen atom, chloro, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, (substituted)phenyl, phenylalkyl, (substituted)phenylalkyl, heteroaryl, (heteroaryl)alkyl or (CH₂)_(p)CO₂R⁵, wherein p=0-4, and R⁵ is as defined below; R³ is a hydrogen atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted)phenylalkyl; R⁴ is a hydrogen atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted)phenylalkyl; R⁵ is a hydrogen atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted)phenylalkyl; A is a natural and unnatural amino acid; B is a hydrogen atom, a deuterium atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, (substituted)phenyl, phenylalkyl, (substituted)phenylalkyl, heteroaryl, (heteroaryl)alkyl, halomethyl, CH₂ZR⁶, CH₂OCO(aryl), CH₂OCO(heteroaryl); or CH₂OPO(R⁷)R⁸ where Z is an oxygen or a sulfur atom; R⁶ is phenyl, substituted phenyl, phenylalkyl, substituted phenylalkyl, heteroaryl, or (heteroaryl)alkyl; and R⁷ and R⁸ are independently selected from a group consisting of alkyl, cycloalkyl, phenyl, substituted phenyl, phenylalkyl, (substituted phenyl) alkyl, and (cycloalkyl) alkyl; and X and Y are independently selected from the group consisting of a hydrogen atom, halo, trihalomethyl, amino, protected amino, an amino salt, mono-substituted amino, di-substituted amino, carboxy, protected carboxy, a carboxylate salt, hydroxy, protected hydroxy, a salt of a hydroxy group, lower alkoxy, lower alkylthio, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, (cycloalkyl)alkyl, substituted (cycloalkyl)alkyl, phenyl, substituted phenyl, phenylalkyl, and (substituted phenyl)alkyl; or a pharmaceutically acceptable salt thereof.
 14. The composition of claim 10, wherein the reagent is a compound of formula 3:

wherein: n is 1 or 2; m is 1 or 2; A is R²CO—, R³—O—CO—, or R⁴SO₂—; a group of the formula:

further wherein: R¹ is a hydrogen atom, alkyl or phenylalkyl; R² is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; R³ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted phenyl)alkyl; R⁴ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; R⁵ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; R⁶ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted phenyl)alkyl; R⁷ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; R⁸ is an amino acid side chain chosen from the group consisting of natural and unnatural amino acids; B is a hydrogen atom, a deuterium atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, (heteroaryl)alkyl, or halomethyl; a group of the formula: —CH₂XR⁹; wherein R⁹ is phenyl, substituted phenyl, phenylalkyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; and X is an oxygen or a sulfur atom; a group of the formula: —CH₂—O—CO-(ARYL); a group of the formula: —CH₂—O—CO-(HETEROARYL); a group of the formula: —CH₂—O—PO(R¹⁰)R¹¹ wherein R¹⁰ and R¹¹ are independently selected from a group consisting of alkyl, cycloalkyl, phenyl, substituted phenyl, phenylalkyl and (substituted phenyl) alkyl; and the pharmaceutically-acceptable salts thereof.
 15. A method for preventing or ameliorating inflammation due to contact of the skin of a mammal with an irritant comprising contacting the skin with a reagent that suppresses the protease activity of at least one member of the interleukin-lbeta-converting enzyme (ICE)/CED-3 family.
 16. The method of claim 15, wherein the irritant is a chemical irritant.
 17. The method of claim 16, wherein the chemical irritant is a cosmetic.
 18. The method of claim 16, wherein the chemical irritant is from a plant.
 19. The method of claim 18, wherein the plant is selected from the group consisting of Poison Ivy, Poison Oak, and Poison Sumac.
 20. The method of claim 15, wherein the irritant is radiation.
 21. The method of claim 20, wherein the radiation is ultraviolet radiation.
 22. The method of claim 15, wherein the reagent suppresses the protease activity in an irreversible manner.
 23. The method of claim 15, wherein the reagent suppresses the protease activity in a reversible manner.
 24. The method of claim 15, wherein the reagent is a compound of formula 1:

wherein: n is 1 or 2; R¹ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, (substituted)phenyl, phenylalkyl, (substituted)phenylalkyl, heteroaryl, (heteroaryl)alkyl or (CH₂)_(m)CO₂R⁴, wherein m=1-4, and R⁴ is as defined below; R² is a hydrogen atom, chloro, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, (substituted)phenyl, phenylalkyl, (substituted)phenylalkyl, heteroaryl, (heteroaryl)alkyl or (CH₂)_(p)CO₂R⁵, wherein p=0-4, and R⁵ is as defined below; R³ is a hydrogen atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted)phenylalkyl; R⁴ is a hydrogen atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted)phenylalkyl; R⁵ is a hydrogen atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted)phenylalkyl; A is a natural and unnatural amino acid; B is a hydrogen atom, a deuterium atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, (substituted)phenyl, phenylalkyl, (substituted)phenylalkyl, heteroaryl, (heteroaryl)alkyl, halomethyl, CH₂ZR⁶, CH₂OCO(aryl), CH₂OCO(heteroaryl); or CH₂OPO(R⁷)R⁸ where Z is an oxygen or a sulftir atom; R⁶ is phenyl, substituted phenyl, phenylalkyl, substituted phenylalkyl, heteroaryl, or (heteroaryl)alkyl; and R⁷ and R⁸ are independently selected from a group consisting of alkyl, cycloalkyl, phenyl, substituted phenyl, phenylalkyl, (substituted phenyl) alkyl, and (cycloalkyl) alkyl; and X and Y are independently selected from the group consisting of a hydrogen atom, halo, trihalomethyl, amino, protected amino, an amino salt, mono-substituted amino, di-substituted amino, carboxy, protected carboxy, a carboxylate salt, hydroxy, protected hydroxy, a salt of a hydroxy group, lower alkoxy, lower alkylthio, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, (cycloalkyl)alkyl, substituted (cycloalkyl)alkyl, phenyl, substituted phenyl, phenylalkyl, and (substituted phenyl)alkyl; or a pharmaceutically acceptable salt thereof.
 25. The method of claim 15, wherein the reagent is a compound of formula 3:

wherein: n is 1 or 2; m is 1 or 2; A is R²CO—, R³—O—CO—, or R⁴SO₂—; a group of the formula:

further wherein: R¹ is a hydrogen atom, alkyl or phenylalkyl; R² is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; R³ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted phenyl)alkyl; R⁴ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; R⁵ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; R⁶ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted phenyl)alkyl; R⁷ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; R⁸ is an amino acid side chain chosen from the group consisting of natural and unnatural amino acids; B is a hydrogen atom, a deuterium atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, (heteroaryl)alkyl, or halomethyl; a group of the formula: —CH₂XR⁹; wherein R⁹ is phenyl, substituted phenyl, phenylalkyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; and X is an oxygen or a sulfur atom; a group of the formula: —CH₂—O—CO-(ARYL); a group of the formula: —CH₂—O—CO-(HETEROARYL); a group of the formula: —CH₂—O-PO(R¹⁰)R¹¹ wherein R¹⁰ and R¹¹ are independently selected from a group consisting of alkyl, cycloalkyl, phenyl, substituted phenyl, phenylalkyl and (substituted phenyl) alkyl; and the pharmaceutically-acceptable salts thereof.
 26. A composition comprising a reagent that suppresses the protease activity of at least one member of the interleukin-l beta-converting enzyme (ICE)/CED-3 family formulated for topical administration for use in preventing or ameliorating inflammation due to skin irritation.
 27. The composition of claim 26, wherein said formulation is selected from a lotion, a cream, a gel, a liquid, a solid, or a semisolid.
 28. The composition of claim 26, wherein the skin irritation is due to contact of the skin with a chemical irritant.
 29. The composition of claim 28, wherein the chemical irritant is a cosmetic or an agent derived from a plant.
 30. The composition of claim 26, wherein the irritant is radiation.
 31. The composition of claim 26, wherein the irritation is due to an insect sting.
 32. The composition of claim 26, wherein the irritation is due to an insect bite.
 33. The composition of claim 26, wherein the irritation is due to tissue damage.
 34. The composition of claim 26, wherein the tissue damage is due to physical trauma or disease.
 35. The composition of claim 33, wherein the tissue (physical trauma or disease) damage is selected from the group consisting of a bum, a scrape, a cut, frostbite, and chemical injury.
 36. The composition of claim 26, wherein the reagent suppresses the protease activity in an irreversible manner.
 37. The composition of claim 26, wherein the reagent suppresses the protease activity in a reversible manner.
 38. The composition of claim 26, wherein the reagent is a compound of formula 1:

wherein: n is 1 or 2; R¹ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, (substituted)phenyl, phenylalkyl, (substituted)phenylalkyl, heteroaryl, (heteroaryl)alkyl or (CH₂)_(m)CO₂R⁴ wherein m=1-4, and R⁴ is as defined below; R² is a hydrogen atom, chloro, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, (substituted)phenyl, phenylalkyl, (substituted)phenylalkyl, heteroaryl, (heteroaryl)alkyl or (CH₂)_(p)CO₂R⁵, wherein p=0-4, and R⁵ is as defined below; R³ is a hydrogen atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted)phenylalkyl; R⁴ is a hydrogen atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted)phenylalkyl; R⁵ is a hydrogen atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted)phenylalkyl; A is a natural and unnatural amino acid; B is a hydrogen atom, a deuterium atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, (substituted)phenyl, phenylalkyl, (substituted)phenylalkyl, heteroaryl, (heteroaryl)alkyl, halomethyl, CH₂ZR⁶, CH₂OCO(aryl), CH₂OCO(heteroaryl); or CH₂OPO(R⁷)R⁸ where Z is an oxygen or a sulfur atom; R⁶ is phenyl, substituted phenyl, phenylalkyl, substituted phenylalkyl, heteroaryl, or (heteroaryl)alkyl; and R⁷ and R⁸ are independently selected from a group consisting of alkyl, cycloalkyl, phenyl, substituted phenyl, phenylalkyl, (substituted phenyl) alkyl, and (cycloalkyl) alkyl; and X and Y are independently selected from the group consisting of a hydrogen atom, halo, trihalomethyl, amino, protected amino, an amino salt, mono-substituted amino, di-substituted amino, carboxy, protected carboxy, a carboxylate salt, hydroxy, protected hydroxy, a salt of a hydroxy group, lower alkoxy, lower alkylthio, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, (cycloalkyl)alkyl, substituted (cycloalkyl)alkyl, phenyl, substituted phenyl, phenylalkyl, and (substituted phenyl)alkyl; or a pharmaceutically acceptable salt thereof.
 39. The composition of claim 26, wherein the reagent is a compound of formula 3:

wherein: n is 1 or 2; m is 1 or 2; A is R²CO—, R³—O—CO—, or R⁴SO₂—; a group of the formula:

further wherein: R¹ is a hydrogen atom, alkyl or phenylalkyl; R² is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; R³ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted phenyl)alkyl; R⁴ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; R⁵ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; R⁶ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted phenyl)alkyl; R⁷ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; R⁸ is an amino acid side chain chosen from the group consisting of natural and unnatural amino acids; B is a hydrogen atom, a deuterium atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, (heteroaryl)alkyl, or halomethyl; a group of the formula: —CH₂XR⁹; wherein R⁹ is phenyl, substituted phenyl, phenylalkyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; and X is an oxygen or a sulfur atom; a group of the formula: —CH₂—O—CO-(ARYL); a group of the formula: —CH₂—O—CO-(HETEROARYL); a group of the formula: —CH₂—O—PO(R¹⁰)R¹¹ wherein R¹⁰ and R¹¹ are independently selected from a group consisting of alkyl, cycloalkyl, phenyl, substituted phenyl, phenylalkyl and (substituted phenyl) alkyl; and the pharmaceutically-acceptable salts thereof.
 40. A method for preventing or ameliorating inflammation due to contact of a tissue of a mammal with an irritant comprising contacting said tissue with a reagent that suppresses the protease activity of at least one member of the interleukin-1 beta-converting enzyme (ICE)/CED-3 family.
 41. The method of claim 40, wherein the irritant is a chemical irritant.
 42. The method of claim 41, wherein the chemical irritant is a cosmetic.
 43. The method of claim 41, wherein the chemical irritant is from a plant.
 44. The method of claim 43, wherein the plant is selected from the group consisting of Poison Ivy, Poison Oak, and Poison Sumac.
 45. The method of claim 40, wherein the irritant is radiation.
 46. The method of claim 45, wherein the radiation is ultraviolet radiation.
 47. The method of claim 40, wherein the irritant is a bacteria.
 48. The method of claim 40, wherein the reagent suppresses the protease activity in an irreversible manner.
 49. The method of claim 40, wherein the reagent suppresses the protease activity in a reversible manner.
 50. The method of claim 40, wherein the reagent is a compound of formula 1:

wherein: n is 1 or 2; R¹ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, (substituted)phenyl, phenylalkyl, (substituted)phenylalkyl, heteroaryl, (heteroaryl)alkyl or (CH₂)_(m)CO₂R⁴, wherein m=1-4, and R⁴ is as defined below; R² is a hydrogen atom, chloro, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, (substituted)phenyl, phenylalkyl, (substituted)phenylalkyl, heteroaryl, (heteroaryl)alkyl or (CH₂)_(p)CO₂R⁵, wherein p=0-4, and R⁵ is as defined below; R³ is a hydrogen atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted)phenylalkyl; R⁴ is a hydrogen atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted)phenylalkyl; R⁵ is a hydrogen atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted)phenylalkyl; A is a natural and unnatural amino acid; B is a hydrogen atom, a deuterium atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, (substituted)phenyl, phenylalkyl, (substituted)phenylalkyl, heteroaryl, (heteroaryl)alkyl, halomethyl, CH₂ZR⁶, CH₂OCO(aryl), CH₂OCO(heteroaryl); or CH₂OPO(R⁷)R⁸ where Z is an oxygen or a sulfur atom; R⁶ is phenyl, substituted phenyl, phenylalkyl, substituted phenylalkyl, heteroaryl, or (heteroaryl)alkyl; and R⁷ and R⁸ are independently selected from a group consisting of alkyl, cycloalkyl, phenyl, substituted phenyl, phenylalkyl, (substituted phenyl) alkyl, and (cycloalkyl) alkyl; and X and Y are independently selected from the group consisting of a hydrogen atom, halo, trihalomethyl, amino, protected amino, an amino salt, mono-substituted amino, di-substituted amino, carboxy, protected carboxy, a carboxylate salt, hydroxy, protected hydroxy, a salt of a hydroxy group, lower alkoxy, lower alkylthio, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, (cycloalkyl)alkyl, substituted (cycloalkyl)alkyl, phenyl, substituted phenyl, phenylalkyl, and (substituted phenyl)alkyl; or a pharmaceutically acceptable salt thereof.
 51. The method of claim 40, wherein the reagent is a compound of formula 3:

wherein: n is 1 or 2; m is 1 or 2; A is R²CO—, R³—O—CO—, or R⁴SO₂—; a group of the formula:

further wherein: R¹ is a hydrogen atom, alkyl or phenylalkyl; R² is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; R³ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted phenyl)alkyl; R⁴ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; R⁵ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; R⁶ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted phenyl)alkyl; R⁷ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; R⁸ is an amino acid side chain chosen from the group consisting of natural and unnatural amino acids; B is a hydrogen atom, a deuterium atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, (heteroaryl)alkyl, or halomethyl; a group of the formula: —CH₂XR⁹; wherein R⁹ is phenyl, substituted phenyl, phenylalkyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; and X is an oxygen or a sulfur atom; a group of the formula: —CH₂—O—CO-(ARYL); a group of the formula: —CH₂—O—CO-(HETEROARYL); a group of the formula:  —CH₂—O—PO(R¹⁰)R¹¹ wherein R¹⁰ and R¹¹ are independently selected from a group consisting of alkyl, cycloalkyl, phenyl, substituted phenyl, phenylalkyl and (substituted phenyl) alkyl; and the pharmaceutically-acceptable salts thereof.
 52. A method for preventing or ameliorating inflammation associated with tissue damage comprising contacting said tissue with a reagent that suppresses the protease activity of at least one member of the interleukin-1 beta-converting enzyme (ICE)/CED-3 family.
 53. The method of claim 52, wherein said tissue damage is due to physical trauma.
 54. The method of claim 52, wherein said tissue damage is due to an autoimmune response.
 55. The method of claim 52, wherein said tissue damage is due to an infectious disease.
 56. The method of claim 52, wherein said tissue damage is due to chronic disease.
 57. The method of claim 52, wherein said tissue damage is spinal or brain trauma.
 58. The method of claim 52, wherein said tissue damage is due to an acid.
 59. The method of claim 52, wherein said tissue damage is due to a base.
 60. The method of claim 52, wherein said tissue damage is due to radiation.
 61. The method of claim 52, wherein the reagent suppresses the protease activity in an irreversible manner.
 62. The method of claim 52, wherein the reagent suppresses the protease activity in a reversible manner.
 63. The method of claim 52, wherein the reagent is a compound of formula 1:

wherein: n is 1 or 2; R¹ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, (substituted)phenyl, phenylalkyl, (substituted)phenylalkyl, heteroaryl, (heteroaryl)alkyl or (CH₂)_(m)CO₂R⁴, wherein m=1-4, and R⁴ is as defined below; R² is a hydrogen atom, chloro, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, (substituted)phenyl, phenylalkyl, (substituted)phenylalkyl, heteroaryl, (heteroaryl)alkyl or (CH₂)_(p)CO₂R⁵, wherein p=0-4, and R⁵ is as defined below; R³ is a hydrogen atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted)phenylalkyl; R⁴ is a hydrogen atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted)phenylalkyl; R⁵ is a hydrogen atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted)phenylalkyl; A is a natural and unnatural amino acid; B is a hydrogen atom, a deuterium atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, (substituted)phenyl, phenylalkyl, (substituted)phenylalkyl, heteroaryl, (heteroaryl)alkyl, halomethyl, CH₂ZR⁶, CH₂OCO(aryl), CH₂OCO(heteroaryl); or CH₂OPO(R⁷)R⁸ where Z is an oxygen or a sulfur atom; R⁶ is phenyl, substituted phenyl, phenylalkyl, substituted phenylalkyl, heteroaryl, or (heteroaryl)alkyl; and R⁷ and R⁸ are independently selected from a group consisting of alkyl, cycloalkyl, phenyl, substituted phenyl, phenylalkyl, (substituted phenyl) alkyl, and (cycloalkyl) alkyl; and X and Y are independently selected from the group consisting of a hydrogen atom, halo, trihalomethyl, amino, protected amino, an amino salt, mono-substituted amino, di-substituted amino, carboxy, protected carboxy, a carboxylate salt, hydroxy, protected hydroxy, a salt of a hydroxy group, lower alkoxy, lower alkylthio, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, (cycloalkyl)alkyl, substituted (cycloalkyl)alkyl, phenyl, substituted phenyl, phenylalkyl, and (substituted phenyl)alkyl; or a pharmaceutically acceptable salt thereof.
 64. The method of claim 52, wherein the reagent is a compound of formula 3:

wherein: n is 1 or 2; m is 1 or 2; A is R²CO—, R³—O—CO—, or R⁴SO₂—; a group of the formula:

further wherein: R¹ is a hydrogen atom, alkyl or phenylalkyl; R² is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; R³ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted phenyl)alkyl; R⁴ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; R⁵ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; R⁶is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted phenyl)alkyl; R⁷ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; R⁸ is an amino acid side chain chosen from the group consisting of natural and unnatural amino acids; B is a hydrogen atom, a deuterium atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, (heteroaryl)alkyl, or halomethyl; a group of the formula: —CH₂XR⁹; wherein R⁹ is phenyl, substituted phenyl, phenylalkyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; and X is an oxygen or a sulfur atom; a group of the formula: —CH₂—O—CO-(ARYL); a group of the formula: —CH₂—O—CO-(HETEROARYL); a group of the formula: —CH₂—O—PO(R¹⁰)R¹¹ wherein R¹⁰ and R¹¹ are independently selected from a group consisting of alkyl, cycloalkyl, phenyl, substituted phenyl, phenylalkyl and (substituted phenyl) alkyl; and the pharmaceutically-acceptable salts thereof.
 65. A composition comprising a reagent that suppresses the protease activity of at least one member of the interleukin-lbeta-converting enzyme (ICE)/CED-3 family and a pharmaceutical, dermatological, or cosmetic carrier formulated for topical application to the skin or mucus membrane of an animal.
 66. The composition of claim 65, wherein said composition ameliorates symptoms associated with an inflammatory response.
 67. The composition of claim 66, wherein said symptoms comprise itching, redness, or swelling.
 68. The composition of claim 65, wherein said composition is useful in decreasing loss of collagen or maintaining skin elasticity and appearance.
 69. The composition of claim 65, wherein the reagent suppresses the protease activity in an irreversible manner.
 70. The composition of claim 65, wherein the reagent suppresses the protease activity in a reversible manner.
 71. The composition of claim 65, wherein the reagent is a compound of formula 1:

wherein: n is 1 or 2; R¹ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, (substituted)phenyl, phenylalkyl, (substituted)phenylalkyl, heteroaryl, (heteroaryl)alkyl or (CH₂)_(m)CO₂R⁴, wherein m=1-4, and R⁴ is as defined below; R² is a hydrogen atom, chloro, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, (substituted)phenyl, phenylalkyl, (substituted)phenylalkyl, heteroaryl, (heteroaryl)alkyl or (CH₂)_(p)CO₂R⁵, wherein p=0-4, and R⁵ is as defined below; R³ is a hydrogen atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted)phenylalkyl; R⁴ is a hydrogen atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted)phenylalkyl; R⁵ is a hydrogen atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted)phenylalkyl; A is a natural and unnatural amino acid; B is a hydrogen atom, a deuterium atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, (substituted)phenyl, phenylalkyl, (substituted)phenylalkyl, heteroaryl, (heteroaryl)alkyl, halomethyl, CH₂ZR⁶, CH₂OCO(aryl), CH₂OCO(heteroaryl); or CH₂OPO(R⁷)R⁸ where Z is an oxygen or a sulfur atom; R⁶ is phenyl, substituted phenyl, phenylalkyl, substituted phenylalkyl, heteroaryl, or (heteroaryl)alkyl; and R⁷ and R⁸ are independently selected from a group consisting of alkyl, cycloalkyl, phenyl, substituted phenyl, phenylalkyl, (substituted phenyl) alkyl, and (cycloalkyl) alkyl; and X and Y are independently selected from the group consisting of a hydrogen atom, halo, trihalomethyl, amino, protected amino, an amino salt, mono-substituted amino, di-substituted amino, carboxy, protected carboxy, a carboxylate salt, hydroxy, protected hydroxy, a salt of a hydroxy group, lower alkoxy, lower alkylthio, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, (cycloalkyl)alkyl, substituted (cycloalkyl)alkyl, phenyl, substituted phenyl, phenylalkyl, and (substituted phenyl)alkyl; or a pharmaceutically acceptable salt thereof.
 72. The composition of claim 65, wherein the reagent is a compound of formula 3:

wherein: n is 1 or 2; m is 1 or 2; A is R²CO—, R³—O—CO—, or R⁴SO₂—; a group of the formula:

further wherein: R¹ is a hydrogen atom, alkyl or phenylalkyl; R² is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; R³ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted phenyl)alkyl; R⁴ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; R⁵ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; R⁶ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted phenyl)alkyl; R⁷ is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; R⁸ is an amino acid side chain chosen from the group consisting of natural and unnatural amino acids; B is a hydrogen atom, a deuterium atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, (heteroaryl)alkyl, or halomethyl; a group of the formula: —CH₂XR⁹; wherein R⁹ is phenyl, substituted phenyl, phenylalkyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; and X is an oxygen or a sulfur atom; a group of the formula: —CH₂—O—CO-(ARYL); a group of the formula: —CH₂—O—CO-(HETEROARYL); a group of the formula:  —CH₂—O-PO(R¹⁰)R¹¹ wherein R¹⁰ and R¹¹ are independently selected from a group consisting of alkyl, cycloalkyl, phenyl, substituted phenyl, phenylalkyl and (substituted phenyl) alkyl; and the pharmaceutically-acceptable salts thereof.
 73. A method for reducing inflammation of a tissue, comprising contacting said tissue with an effective amount of a reagent that suppresses the protease activity of at least one member of the interleukin-lbeta-converting enzyme (ICE)/CED-3 family, thereby reducing inflammation of said tissue.
 74. The method of claim 73, wherein said tissue is skin.
 75. The method of claim 74, wherein said tissue inflammation is due to trauma, sunburn, eczema, contact allergy, dermatitis, psoriasis, erysipelas, acne, ingrown nails, cuts, burns, insect bites, insect stings, or pruritus.
 76. The method of claim 73, wherein said tissue is mucosa.
 77. The method of claim 73, wherein said tissue inflammation is due to vaginitis, hemorrhoids, conjunctivitis, periodontitis, wisdom tooth eruption, teeth extraction, gingivitis, periodontal abscesses, or prosthesis. 